Thienopyridine compounds, their production and use

ABSTRACT

The compound of the present invention possesses excellent gonadotropin-releasing hormone antagonizing activity, and is useful for preventing or treating sex hormone-dependent diseases, e.g., sex hormone-dependent cancers (e.g., prostatic cancer, uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy, hysteromyoma, endometriosis, precocious puberty, amenorrhea syndrome, multilocular ovary syndrome, pimples etc, or as a pregnancy regulator (e.g., contraceptive), infertility remedy or menstruation regulator.

TECHNICAL FIELD

1. The present invention relates to thieno[2,3-b]pyridine derivativesexhibiting gonadotropin releasing hormone (GnRH) antagonizing activity,their production and use.

BACKGROUND ART

2. The secretion of hypophysial anterior lobe hormone is regulated bythe peripheral hormone secreted by each target organ and thesecretion-promoting or secretion-suppressing hormone secreted by thehypothalamus, which is the center superior to the hypophysial anteriorlobe, and this group of hormones hereinafter generically referred to ashypothalamic hormone in this specification. To date, nine hypothalamichormones have been identified, for example, thyroid-stimulatinghormone-releasing hormone (TRH), and gonadotropin releasing hormone[GnRH, also known as luteinizing hormone releasing hormone (LH-RH)],etc. It is conjectured that these hypothalamic hormones exhibit theirhormone actions etc. via receptors assumed to be present in thehypophysial anterior lobe, and analyses of receptor genes specific tothese hormones, including humans, are ongoing. Antagonists or agoniststhat act specifically and selectively on these receptors would thereforeregulate the action of hypothalamic hormones and hence regulate thesecretion of hypophysial anterior lobe hormone. As a result, suchantagonists or agonists are expected to prevent or treat diseasesdepending on these hypophysial anterior lobe hormone.

3. Known compounds possessing GnRH-antagonizing activity includeGnRH-derived linear peptides (U.S. Pat. No. 5,140,009 and U.S. Pat. No.5,171,835), a cyclic hexapeptide derivative (JP-A-61-191698), a bicyclicpeptide derivative [Journal of Medicinal Chemistry, Vol. 36, pp.3265-3273 (1993)], and so forth. Non-peptide compounds possessingGnRH-antagonizing activity include compounds described in WO 95/28405,WO 97/14697, WO 97/14682, WO 97/41126 and so forth.

4. Peptide compounds pose a large number of problems to be resolved withrespect to oral absorbability, dosage form, dose volume, drug stability,sustained action, metabolic stability etc. There is strong demand for anoral GnRH antagonist, especially one based on a non-peptide compound,that has excellent therapeutic effect on hormone-dependent cancers,e.g., prostatic cancer, endometriosis, precocious puberty etc., and thatdoes not show transient hypophysial-gonadotropic action (acute action).

BRIEF DESCRIPTION OF DRAWINGS

5.FIG. 1 is a diagrammatic representation of the ameliorating effect ofsuppression of plasma LH concentrations in castrated monkeys after oraladministration of the compound of the following Example 1-2, in which .. . ▪ . . . represents control (1) (vehicle), . . . ♦ . . . representscontrol (2) (vehicle), . . . □ . . . represents control (3) (vehicle),-Δ- represents compound (1) [the test animal administered the compoundof Ex. No. 1-2] and -▴- represents compound (2) [the test animaladministered the compound of Ex. No. 1-2].

DISCLOSURE OF INVENTION

6. The present inventors produced various thienopyridine derivatives,investigated their actions, and found that some compounds possessexcellent GnRH-antagonizing activity. The inventors have conductedfurther investigation based on this finding, and developed the presentinvention.

7. Accordingly, the present invention relates to:

8. [1] a compound of the formula:

9. wherein R¹ represents a C₁₇ alkyl group which may be substituted, aC₃₋₇ cycloalkyl group which may be substituted, a C₁₋₆ alkoxyamino groupwhich may be substituted or a hydroxyamino group which may besubstituted; and

10. R² represents a C₁₋₇ alkyl group which may be substituted or aphenyl group which may be substituted;

11. when R¹ is an unsubstituted C₁₋₇ alkyl group, then R² is asubstituted C₁₋₇ alkyl group or a substituted phenyl group, or a saltthereof [hereinafter sometimes referred to briefly as compound (I)];

12. [2] a compound of the above (1) or a salt thereof, wherein R¹ is (1)a C₁₋₇ alkyl group which may be substituted by 1 to 5 substituentsselected from the group consisting of (i) hydroxy, (ii) C₁₋₇ acyloxy,(iii) amino which may be substituted by 1 or 2 substituents selectedfrom the group consisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl,C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy whichmay be substituted by 1 to 3 substituents selected from the groupconsisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆alkoxy-carbonyl,

13. (2) a C₃₋₇ cycloalkyl group which may be substituted by 1 to 3substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl,

14. (3) a C₁₋₆ alkoxyamino group which may be substituted by 1 to 5substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl, or

15. (4) a hydroxyamino group which may be substituted by 1 or 2substituents selected from the group consisting of (i) C₁₋₇ acyloxy,(ii) amino which may be substituted by 1 or 2 substituents selected fromthe group consisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iii) C₁₋₁₀ alkoxy which may besubstituted by 1 to 3 substituents selected from the group consisting ofC₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (iv) C₁₋₆ alkyl; and

16. R² is (1) a C₁₋₇ alkyl group which may be substituted by 1 to 5substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl, or

17. (2) a phenyl group which may be substituted by 1 to 5 substituentsselected from the group consisting of halogen, C₁₋₃ alkyl and C₁₋₃alkoxy;

18. [3] a compound of the above (1) or a salt thereof, wherein R¹ is aC₁₋₇ alkyl group which may be substituted or a C₃₋₇ cycloalkyl groupwhich may be substituted;

19. [4] a compound of the above (1) or a salt thereof, wherein R¹ is asubstituted branched C₃₋₇ alkyl group or a substituted C₃₋₇ cycloalkylgroup;

20. [5] a compound of the above (1) or a salt thereof, wherein R¹ is aC₁₋₇ alkyl group substituted by hydroxy or a C₃₋₇ cycloalkyl groupsubstituted by hydroxy;

21. [6] a compound of the above (1) or a salt thereof, wherein R¹ is asubstituted C₃₋₇ cycloalkyl group;

22. [7] a compound of the above (1) or a salt thereof, wherein R¹ is acyclopropyl group which may be substituted by hydroxy;

23. [8] a compound of the above (1) or a salt thereof, wherein R² is abranched C₃₋₇ alkyl group which may be substituted;

24. [9] a compound of the above (1) or a salt thereof, wherein R² is aphenyl group which may be substituted;

25. [10] a compound of the above (1) or a salt thereof, wherein R² is aphenyl group;

26. [11] a compound of the above (1) or a salt thereof, wherein R¹ is aC₃₋₇ cycloalkyl group and R² is a C₁₋₆ alkyl group;

27. [12] a compound of the above (1) or a salt thereof, wherein R¹ is(1) a C₁₋₄ alkyl group substituted by 1 or 2 hydroxy, (2) a C₃₋₇cycloalkyl group substituted by hydroxy, or (3) a C₁₋₃ alkoxyaminogroup; and R² is an isopropyl group or a phenyl group;

28. [13] a compound of the above (1) or a salt thereof, wherein R¹ is(1) a C₁₋₇ alkyl group which may be substituted by 1 or 2 substituentsselected from the group consisting of hydroxy, C₁₋₃ alkyl-carbonyloxy,amino, benzyloxycarbonylamino, C₁₋₃ alkoxy, C₁₋₃ alkoxy-C₁₋₃ alkoxy andC₁₋₃ alkoxy-carbonyl, (2) a C₃₋₇ cycloalkyl group which may besubstituted by a hydroxy or a C₁₋₃ alkyl-carbonyloxy, or (3) a C₁₋₃alkoxyamino group; and

29. R² is (1) an isopropyl group which may be substituted by a hydroxyor (2) a phenyl group;

30. [14]3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridine or a salt thereof;

31. [15]3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridineor a salt thereof;

32. [16]3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine or a salt thereof;

33. [17]3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b] pyridine or a salt thereof;

34. [18] a process for producing a compound of the above [1] or a saltthereof, which comprises reacting a compound of the formula:

35. wherein R² represents a C₁₋₇ alkyl group which may be substituted ora phenyl group which may be substituted, or a salt thereof [hereinaftersometimes referred to briefly as compound (II)], with

36. i) a compound of the formula:

R^(1a)COOH

37. wherein R^(1a) represents a C₁₋₇ alkyl group which may besubstituted or a C₃₋₇ cycloalkyl group which may be substituted, or asalt thereof or a reactive derivative thereof; or

38. ii) carbonyldiimidazole, phosgene or a chloroformate, followed byreacting with a compound of the formula:

R^(1b)H

39. wherein R^(1b) represents a C₁₋₆ alkoxyamino group which may besubstituted or a hydroxyamino group which may be substituted, or a saltthereof;

40. [19] a process for producing a compound of the above [3] or a saltthereof, which comprises reacting compound (II) with a compound of theformula:

R^(1a)COOH

41. wherein R^(1a) represents a C₁₋₇ alkyl group which may besubstituted or a C₃₋₇ cycloalkyl group which may be substituted, or asalt thereof or a reactive derivative thereof;

42. [20] a pharmaceutical composition which comprises a compound of theabove (1) or a salt thereof;

43. [21] a pharmaceutical composition of the above (20) which is forantagonizing gonadotropin-releasing hormone;

44. [22] a pharmaceutical composition of the above (21) which is forpreventing or treating a sex hormone dependent disease;

45. [23] a method for antagonizing gonadotropin-releasing hormone in amammal in need thereof which comprises administeringto saidmammal aneffective amount of acompound of the above (1) or a salt thereof with apharmaceutically acceptable excipient, carrier or diluent;

46. [24] use of a compound of the above (1) or a salt thereof formanufacturing a pharmaceutical composition for antagonizinggonadotropin-releasing hormone, and so forth.

47. Each symbol in the above formulae is hereinafter described in moredetail.

48. The “C₁₋₇ alkyl group” of the “C₁₋₇ alkyl group which may besubstituted” for R¹ includes, for example, a straight-chain C₁₋₇ alkylgroup such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, etc.;a branched C₃₋₇ alkyl group such as isopropyl, isobutyl, sec-butyl,tert-butyl, isopentyl, neopentyl, etc., and so forth. Among others,preferred is a branched C₃₋₇ alkyl group. More preferred is isopropyl.

49. The “substituents” of the “C₁₋₇ alkyl group which may besubstituted” for R¹ include, for example, (i) hydroxy, (ii) C₁₋₇ acyloxy(e.g., C₁₋₆ alkyl-carbonyloxy such as acetoxy, propionyloxy, etc.;benzoyloxy etc.), (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl(e.g., methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl, etc.),benzyloxycarbonyl, C₁₋₃ acyl (e.g., C₁₋₂ alkyl-carbonyl such as acetyl,propionyl, etc.), C₁₋₃ alkylsulfonyl (e.g., methanesulfonyl etc.) andC₁₋₃ alkyl (e.g., methyl, ethyl, etc.), etc. [e.g.; amino,methoxycarbonylamino, ethoxycarbonylamino,tert-butoxycarbonylbenzyloxycarbonylamino, acetylamino,methanesulfonylamino, methylamino, dimethylamino, etc.), (iv) C₁₋₁₀(preferably C₁₋₄) alkoxy which may be substituted by 1 to 3 substituentsselected from the group consisting of C₃₋₇ cycloalkyloxycarbonyl (e.g.,cyclohexyloxycarbonyloxy, etc.) and C₁₋₃ alkoxy (e.g., methoxy, ethoxy,etc.) [e.g.; methoxy, ethoxy, propoxy, tert-butoxy,cyclohexyloxycarbonyloxy-1-ethoxy, methoxymethoxy, ethoxymethoxy, etc.],(v) C₁₋₆ alkoxycarbonyl (e.g., methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, etc.), and so forth. Among others, preferred ishydroxy.

50. The “C₁₋₇ alkyl group” may have 1 to 5, preferably 1 to 3substituents as mentioned above at possible positions and, when thenumber of substituents is two or more, those substituents may be thesame as or different from one another.

51. The “C₃₋₇ cycloalkyl group” of the “C₃₋₇ cycloalkyl group which maybe substituted” for R¹ includes, for example, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, etc. Among others, preferred iscyclopropyl.

52. The “substituents” of the “C₃₋₇ cycloalkyl group which may besubstituted” for R¹ are the same as those mentioned above for the“substituents” of the “C₁₋₇ alkyl group which may be substituted” forR¹. The number of substituents is 1 to 3. When the number ofsubstituents is two or more, those substituents may be the same as ordifferent from one another.

53. The “C₁₋₆ alkoxyamino group” of the “C₁₋₆ alkoxyamino group whichmay be substituted” for R¹ includes, for example, mono- or di-C₁₋₆alkoxyamino (e.g., methoxyamino, ethoxyamino, dimethoxyamino,diethoxyamino, ethoxymethoxyamino, etc.), etc. Among others, preferredis mono-C₁₋₃ alkoxyamino (e.g., methoxyamino, etc.).

54. The “substituents” of the “C₁₋₆ alkoxyamino group which may besubstituted” for R¹ and their number are the same as those mentionedabove for the “substituents” of the “C₁₋₇ alkyl group which may besubstituted” for R¹. When the number of substituents is two or more,those substituents may be the same as or different from one another. The“C₁₋₆ alkoxy” or the “nitrogen atom of an amino group” of the C₁₋₆alkoxyamino group may be substituted by the above “substituents”.

55. Such “C₁₋₆ alkoxyamino group which may be substituted” isexemplified by methoxyamino, N-methyl-N-methoxyamino,N-ethyl-N-methoxyamino, ethoxyamino, dimethoxyamino, diethoxyamino,ethoxymethoxyamino, etc. Preferred is C₁₋₃ alkoxyamino, N—C₁₋₃alkyl-N—C₁₋₃ alkoxyamino, etc.

56. The “substituents” of the “hydroxyamino group which may besubstituted” for R¹ may be located on the “hydroxy group” of thehydroxyamino group or the “nitrogen atom of an amino group” of thehydroxyamino group. Such “substituents” on the “hydroxy group” include,for example, (i) C₁₋₇ acyloxy (e.g., C₁₋₆ alkyl-carbonyloxysuch asacetoxy, propionyloxy, etc.; benzoyloxy etc.), (ii) amino which may besubstituted by 1 or 2 substituents selected from the group consisting ofC₁₋₆ alkoxy-carbonyl (e.g., methoxycarbonyl, ethoxycarbonyl,tert-butoxycarbonyl, etc.), benzyloxycarbonyl, C₁₋₃ acyl (e.g., C₁₋₂alkyl-carbonyl such as acetyl, propionyl, etc.), C₁₋₃ alkylsulfonyl(e.g., methanesulfonyl etc.) and C₁₋₃ alkyl (e.g., methyl, ethyl, etc.),etc. [e.g.; amino, methoxycarbonylamino, ethoxycarbonylamino,tert-butoxycarbonylbenzyloxycarbonylamino, acetylamino,methanesulfonylamino, methylamino, dimethylamino, etc.], (iii) C₁₋₁₀(preferably C₁₋₄) alkoxy which may be substituted by 1 to 3 substituentsselected from the group consisting of C₃₋₇ cycloalkyloxycarbonyl (e.g.,cyclohexyloxycarbonyloxy, etc.) and C₁₋₃ alkoxy (e.g., methoxy, ethoxy,etc.) [e.g.; methoxy, ethoxy, propoxy, tert-butoxy,cyclohexyloxycarbonyloxy-1-ethoxy, methoxymethoxy, ethoxymethoxy, etc.],and so forth. The number of substituents is 1 to 5, preferably 1 to 3.When the number of substituents is two or more, those substituents maybe the same as or different from one another. Such “substituents” on the“nitrogen atom of the amino group” include, for example, (1) each groupas described in the above (i) to (iii) and (2) C₁₋₆ alkyl (e.g., methyl,ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl,pentyl, isopentyl, neopentyl, hexyl, etc. The number of substituents is1 to 5, preferably 1 to 3. When the number of substituents is two ormore, those substituents may be the same as or different from oneanother.

57. Preferable examples of the “hydroxyamino group which may besubstituted” include N—C₁₋₆ alkyl-N-hydroxyamino (e.g.,N-methyl-N-hydroxyamino, N-ethyl-N-hydroxyamino, etc.) and so forth.More preferred is N—C₁₋₃ alkyl-N-hydroxyamino, etc.

58. The “C₁₋₇ alkyl group” of the “C₁₋₇ alkyl group which may besubstituted” for R² includes, for example, a straight-chain or branchedC₁₋₇ alkyl group such as methyl, ethyl, propyl, isopropyl, butyl,isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl,heptyl, etc. Among others, preferred is C₁₋₃ alkyl (e.g., methyl, ethyl,propyl, isopropyl, etc. More preferred is isopropyl.

59. The “substituents” of the “C₁₋₇ alkyl group which may besubstituted” for R² and their number are the same as those mentionedabove for the “substituents” of the “C₁₋₇ alkyl group which may besubstituted” for R¹. When the number of substituents is two or more,those substituents may be the same as or different from one another.

60. The “substituents” of the “phenyl group which may be substituted”for R² includes, for example, halogen (e.g., fluoro, chloro, bromo,iodo, etc.), C₁₋₃ alkyl (e.g., methyl, ethyl, propyl, isopropyl, etc.),C₁₋₃ alkoxy (e.g., methoxy, ethoxy, propoxy, isopropoxy, etc.), etc.Among others, preferred is halogen, more preferred is fluoro.

61. The “phenyl group” may have 1 to 5, preferably 1 to 3 substituentsas mentioned above at possible positions and, when the number ofsubstituents is two or more, those substituents may be the same as ordifferent from one another.

62. R¹ is preferably a substituted branched C₃₋₇ alkyl group or asubstituted C₃₋₇ cycloalkyl group, more preferably a C₁₋₇ alkyl groupsubstituted by hydroxy or a C₃₋₇ cycloalkyl group substituted byhydroxy. Among others, preferred is a substituted C₃₋₇ cycloalkyl group.Also, a C₁₋₃ alkyl group which may be substituted by hydroxy, a C₃₋₇cycloalkyl group which may be substituted by a hydroxy, mono-C₁₋₃alkoxyamino, N—C₁₋₃ alkyl-N-hydroxyamino, hydroxyamino, is preferred.Especially preferably R¹ is (i) cyclopropyl which may be substituted bya hydroxy or (ii) methoxyamino, etc. Most preferred is cyclopropyl whichmay be substituted by a hydroxy group.

63. R² is preferably a C₁₋₇ alkyl group which may be substituted. Morepreferred is a C₁₋₃ alkyl group which may be substituted by a hydroxygroup. Especially preferred is isopropyl. Phenyl is also preferred.

64. Preferable examples of compound (I) include a compound wherein, R¹is a C₁₋₃ alkyl group which may be substituted by a hydroxy group, aC₃₋₇ cycloalkyl group which may be substituted by a hydroxy group or amono-C₁₋₃ alkoxyamino group; and

65. R² is a C₁₋₃ alkyl group, or a salt thereof.

66. More preferred is a compound wherein R¹ is (1) a C₁₋₄ alkyl groupsubstituted by 1 or 2 hydroxy, (2) a C₃₋₇ cycloalkyl group substitutedby hydroxy, or (3) a C₁₋₃ alkoxyamino group; and

67. R² is an isopropyl group or a phenyl group, or a salt theteof.

68. A compound wherein R¹ is (1) a C₁₋₇ alkyl group which may besubstituted by 1 or 2 substituents selected from the group consisting ofhydroxy, C₁₋₃ alkyl-carbonyloxy, amino, benzyloxycarbonylamino, C₁₋₃alkoxy, C₁₋₃ alkoxy-C₁₋₃ alkoxy and C₁₋₃ alkoxy-carbonyl, (2) a C₃₋₇cycloalkyl group which may be substituted by a hydroxy group or a C₁₋₃alkylcarbonyloxy, or (3) a C₁₋₃ alkoxyamino group; and

69. R² is (1) an isopropyl group which may be substituted by a hydroxygroup or (2) a phenyl group, or salt thereof is also preferred.

70. As compound (I), concretely mentioned are

71.3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-bipyridineor a salt thereof,

72.5-benzoyl-3-(N-benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxo-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]thieno[2,3-b]pyridine,

73.5-(4-fluorobenzoyl)-3-(N-benzyl-N-methylaminomethyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxo-2-(4-cyclopropanecarbonylaminophenyl)thieno[2,3-b]pyridine,

74.3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridine,

75.3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-N′-methoxyureidophenyl)-4-oxothieno[2,3-b]pyridine,

76.3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine,

77.(R)-4,7-dihydro-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

78.4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

79.4,7-dihydro-2-[4-(3-hydroxy-3-methylbutyrylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

80.(R)-4,7-dihydro-2-[4-(2,3-dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine,

81.3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine, and salts thereof.

82. Compound (I) can be produced in any per se known manner, forexample, according to the methods disclosed in WO 95/28405 or analogousmethods thereto. Concretely mentioned are the following Productionmethod 1 and Production method 2.

83. Production Method 1

84. Compound (I) is produced by reacting compound (II) with a compoundof the formula: R^(1a)COOH wherein R^(1a) represents a C₁₋₇ alkyl groupwhich may be substituted or a C₃₋₇ cycloalkyl group which may besubstituted, or a salt thereof or a reactive derivative thereof[hereinafter sometimes referred to briefly as compound (III)].

85. The “C₁₋₇ alkyl group which may be substituted” and the “C₃₋₇cycloalkyl group which may be substituted” for R^(1a) are the same asthose mentioned above for the “C₁₋₇ alkyl group which may besubstituted” and the “C₃₋₇cycloalkyl group which may be substituted” forR¹, respectively.

86. The “reactive derivative” of the “compound of the formula:R^(1a)COOH, or a salt thereof or a reactive derivative thereof”includes, for example, a compound of the formula:

R^(1a)COY

87. wherein Y represents halogen and R^(1a) is same as defined above.

88. The amount of compound (III) to be reacted is about 1 to 3 mol,relative to one mol of compound (II).

89. This reaction is also carried out in the presence of a base. The“base” is exemplified by inorganic bases such as sodium carbonate,sodium hydrogen carbonate, potassium carbonate, potassium hydrogencarbonate, sodium hydroxide, potassium hydroxide and thallium hydroxide,and organic bases such as triethylamine and pyridine, etc.

90. The amount of the “base” is about 1 to 10 mol, preferably about 2 to5 mol, relative to one mol of compound (II).

91. In the case where a compound of the formula: R^(1a)COOH or a saltthereof is used, a condensing reagent which is generally used in peptidechemistry such as benzotroazol-1-yloxytripyrrolidinophosphoniumhexafluorophosphate (PyBop) may be employed to form an amide bond.

92. This reaction is advantageously carried out in a solvent inert tothe reaction. Examples of the solvent include ethers (e.g., ethyl ether,dioxane, dimethoxyethane, tetrahydrofuran, etc.), aromatic hydrocarbons(e.g., benzene, toluene, etc.), amides (e.g., dimethylformamide,dimethylacetamide, etc.), halogenated hydrocarbons (e.g., chloroform,dichloromethane, etc.), and so forth.

93. The reaction temperature is usually about 0 to 150° C., preferablyroom temperature(about 15 to 25° C.). The reaction time is usually about1 to 12 hours.

94. Compound (II) can be produced in any per se known manner, forexample, by the methods disclosed in WO 95/28405 or analogous methodsthereto.

95. Compound (I) wherein R¹ is a C₁₋₆ alkoxyamino group which may besubstituted or a hydroxyamino group which may be substituted can also beproduced in according to the method of the following Production method2.

96. Production Method 2

97. Compound (I) is produced by reacting compound (II) withcarbonyldiimidazole (N,N′-carbonyldiimidazole; CDI), phosgene (monomer,dimer or trimer) or a chloroformate, followed by reacting with acompound of the formula: R^(1b)H wherein R^(1b) represents a C₁₋₆alkoxyamino group which may be substituted or a hydroxyamino group whichmay be substituted, or a salt thereof [hereinafter sometimes referred tobriefly as compound (IV)].

98. The “chloroformate” includes, for example, a compound of theformula: Cl—COOY′ wherein Y′ represents a C₁₋₆ alkyl group, such aschloroformate ethyl, etc.

99. The “C₁₋₆ alkoxyamino group which may be substituted” and the“hydroxyamino group which may be substituted” for R^(1b) are the same asthose mentioned above for the “C₁₋₆ alkoxyamino group which may besubstituted” and the “hydroxyamino group which may be substituted” forR¹, respectively.

100. In the reaction of compound (II) with carbonyldiimidazole, phosgeneor chloroformates, carbonyldiimidazole, phosgene or chloroformates isused in amount of about 1 to 3 mol, relative to one mol of compound(II).

101. This reaction is advantageously carried out in a solvent inert tothe reaction. Examples of the solvent include ethers (e.g., ethyl ether,dioxane, dimethoxyethane, tetrahydrofuran, etc.) , aromatic hydrocarbons(e.g., benzene, toluene, etc.), amides (e.g., dimethylformamide,dimethylacetamide,etc.), halogenated hydrocarbons (e.g., chloroform,dichloromethane, etc.), and so forth.

102. The reaction temperature is usually about 0 to 150° C., preferablyroom temperature (about 15 to 25° C.). The reaction time is usuallyabout 1 to 36 hours.

103. This reaction is also carried out in the presence of a base. The“base” is exemplified by inorganic bases such as sodium carbonate,sodium hydrogen carbonate, potassium carbonate, potassium hydrogencarbonate, sodium hydroxide, potassium hydroxide and thallium hydroxide,and organic bases such as triethylamine and pyridine, etc.

104. The amount of the “base” is about 2 to 20 mol, preferably about 5to 12 mol, relative to one mol of compound (II).

105. The following reaction with compound (IV) can be carried out in thesame condition of the above reaction of compound (II) withcarbonyldiimidazole, phosgene or a chloroformate. The amount of compound(IV) is about 2 to 20 mol, preferably about 5 to 10 mol, relative to onemol of compound (II).

106. The reaction temperature is usually about 0 to 150° C., preferablyroom temperature (about 15 to 25° C.). The reaction time is usuallyabout 1 to 6 hours.

107. Compound (I) of the present invention may be isolated and purifiedby ordinary means of separation such as recrystallization, distillationand chromatography, etc. When the compound of the formula (I) isobtained in free form, it can be converted to a salt by per se knownmethods or analogous thereto. When compound (I) is obtained in saltform, it can be converted to the free form or another salt by per seknown methods or analogous thereto.

108. Salts of compound (I) are preferably physiologically acceptableacid addition salts. Such salts include, for example, salts withinorganic acids (e.g., hydrochloric acid, hydrobromic acid, nitric acid,sulfuric acid, phosphoric acid) and physiologically acceptable acidaddition salts with organic acids (e.g., formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleicacid, citric acid, succinic acid, malic acid, methanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid). When compound (I) has anacidic group, it nay form a physiologically acceptable salt with aninorganic base (e.g., alkali metals such as sodium, potassium, calciumand magnesium, alkaline earth metals, ammonia) or an organic base (e.g.,trimethylamine, triethylamine, pyridine, picoline, ethanolamine,diethanolamine, triethanolamine, dicyclohexylamine,N,N′-dibenzylethylenediamine).

109. Compound (I) may be a hydrate or a non-hydrate. The hydrate isexemplified by monohydrate, sesquihydrate and dehydrate.

110. When compound (I) is obtained as a mixture (racemate) of opticallyactive configurations, it can be resolved into the (R)- and (S)- formsby the conventional optical resolution techniques.

111. Compound (I) of the present invention (hereinafter also referred toas “compound of the present invention”) possesses excellentGnRH-antagonizing activity and low toxicity. In addition, it isexcellent in oral absorbability, action sustainability, stability andpharmacokinetics. Furthermore, it can be easily produced. The compoundof the present invention can therefore be safely used in a mammal (e.g.,human, monkey, bovine, horse, dog, cat, rabbit, rat, mouse) for thepreventing and/or treating diseases depending on male or femalehormones, diseases due to excess of these hormone, etc., by suppressinggonadotropin secretion by its GnRH receptor-antagonizing action tocontrol blood sex hormone concentrations.

112. For example, the compound of the present invention is useful forpreventing and/or treating sex hormone-dependent cancers (e.g.,prostatic cancer, uterine cancer, breast cancer, pituitary tumor, etc.),prostatic hypertrophy, hysteromyoma, endometriosis, precocious puberty,amenorrhea, premenstrual syndrome, multilocular ovary syndrome, pimplesetc. The compound of the present invention is also useful for theregulation of reproduction in males and females (e.g., pregnancyregulators, menstruation cycle regulators, etc.). The compound of thepresent invention also be used as a male or female contraceptive, or asa female ovulation inducer. Based on its rebound effect afterwithdrawal, the compound of the present invention can be used to treatinfertility.

113. In addition, the compound of the present invention is useful forregulation of animal estrous, improvement of meat quality and promotionof animal growth in the field of animal husbandry. The compound of thepresent invention is also useful as a fish spawning promoter.

114. Although the compound of the present invention can be used alone,it is effective to use in combination with a steroidal or non-steroidalanti-androgen agent or anti-estrogen agent. The compound of the presentinvention can also be used to suppress the transient rise in bloodtestosterone concentration (flare phenomenon) observed in administrationof a super-agonist such as Leuprorelin acetate. The compound of thepresent invention can be used in combination with a super-agonist suchas leuprorelin acetate, gonadrelin, buserelin, triptorelin, goserelin,nafarelin, histrelin, deslorelin, meterelin, lecirelin, and so forth.Among others, preferred is leuprorelin acetate. The compound of thepresent invention also may be used with a chemotherapeutic agent forcancer. A preferred example of such combination is the compound of thepresent invention in combination with chemotherapeutic agents such asifosfamide, UTF, adriamycin, peplomycin and cisplatin for prostaticcancer. For breast cancer, the compound of the present invention can beused with chemotherapeutic agents such as cyclophosphamide, 5-FU, UFT,methotrexate, adriamycin, mitomycin C and mitoxantrone.

115. When the compound of the present invention is used for preventingand/or treating (as a prophylactic and/or therapeutic agent for) theabove-mentioned diseases or used in the filed of animal husbandry orfishery, it can be administered orally or non-orally, as formulated witha pharmaceutically acceptable carrier, normally in the form of solidpreparations such as tablets, capsules, granules and powders for oraladministration, or in the form of intravenous, subcutaneous,intramuscular or other injections, suppositories or sublingual tabletsfor non-oral administration. It may also be sublingually,subcutaneously, intramuscularly or otherwise administered in the form ofsustained-release preparations of sublingual tablets, microcapsules etc.Depending on symptom severity; subject age, sex, weight and sensitivity;duration and intervals of administration; property, dispensing and kindof pharmaceutical preparation; kind of active ingredient etc., dailydose is not subject to limitation. For use in the treatment of theabove-described sex hormone-dependent cancers (e.g., prostatic cancer,uterine cancer, breast cancer, pituitary tumor), prostatic hypertrophy,hysteromyoma, endometriosis, precocious puberty etc., daily dose isnormally about 0.01 to 30 mg, preferably about 0.02 to 10 mg, and morepreferably 0.1 to 10 mg, per kg weight of mammal, normally in 1 to 4divided dosages.

116. The above doses are applicable to the use of the compound of thepresent invention in the filed of animal husbandry or fishery. Dailydose is about 0.01 to 30 mg, preferably about 0.1 to 10 mg, per kgweight of subject organism, normally in 1 to 3 divided dosages.

117. In the pharmaceutical composition of the present invention, theamount of compound (I) is 0.01 to 100% by weight or so of the totalweight of the composition.

118. Pharmaceutically acceptable carriers are various organic orinorganic carrier substances in common use as pharmaceutical materials,including excipients, lubricants, binders and disintegrants for solidpreparations, and solvents, dissolution aids, suspending agents,isotonizing agents, buffers and soothing agents for liquid preparations.Other pharmaceutical additives such as preservatives, antioxidants,coloring agents and sweetening agents may be used as necessary.

119. Preferable excipients include, for example, lactose, sucrose,D-mannitol, starch, crystalline cellulose and light silicic anhydride.Preferable lubricants include, for example, magnesium stearate, calciumstearate, talc and colloidal silica. Preferable binders include, forexample, crystalline cellulose, sucrose, D-mannitol, dextrin,hydroxypropyl cellulose, hydroxypropylmethyl cellulose andpolyvinylpyrrolidone. Preferable disintegrants include, for example,starch, carboxymethyl cellulose, carboxymethyl cellulose calcium,crosslinked carmellose sodium and carboxymethyl starch sodium.Preferable solvents include, for example, water for injection, alcohol,propylene glycol, macrogol, sesame oil and corn oil. Preferabledissolution aids include, for example, polyethylene glycol, propyleneglycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane,cholesterol, triethanolamine, sodium carbonate and sodium citrate.Preferable suspending agents include, for example, surfactants such asstearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionicacid, lecithin, benzalkonium chloride, benzethonium chloride andmonostearic glycerol; and hydrophilic polymers such as polyvinylalcohol, polyvinylpyrrolidone, carboxymethyl cellulose sodium, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose andhydroxypropyl cellulose. Preferable isotonizing agents include, forexample, sodium chloride, glycerol and D-mannitol. Preferable buffersinclude, for example, buffer solutions of phosphates, acetates,carbonates, citrates etc. Preferable soothing agents include, forexample, benzyl alcohol. Preferable preservatives include, for example,paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethylalcohol, dehydroacetic acidand sorbic acid. Preferable antioxidantsinclude, for example, sulfites and ascorbic acid.

120. By adding suspending agents, dissolution aids, stabilizers,isotonizing agents, preservatives, and so forth, the compound of thepresent invention can be prepared as an intravenous, subcutaneous orintramuscular injection by a commonly known method. In such cases, thecompound of the present invention can be freeze-dried as necessary by acommonly known method. In administration to humans, for example, thecompound of the present invention can be safely administered orally ornon-orally as such or as a pharmaceutical composition prepared by mixingit with a pharmacologically acceptable carrier, excipient and diluentselected as appropriate.

121. Such pharmaceutical compositions include oral preparations (e.g.,powders, granules, capsules, tablets), injections, drip infusions,external preparations (e.g., nasal preparations, transdermalpreparations) and suppositories (e.g., rectal suppositories, vaginalsuppositories).

122. These preparations can be produced by commonly known methods incommon use for pharmaceutical making processes.

123. An injection can be produced by, for example, preparing thecompound of the present invention as an aqueous injection along with adispersing agent (e.g., Tween 80, produced by Atlas Powder Company, USA,HCO 60, produced by Nikko Chemicals Co., Ltd., polyethylene glycol,carboxymethyl cellulose, sodium alginate), a preservative (e.g., methylparaben, propyl paraben, benzyl alcohol), an isotonizing agent (e.g.,sodium chloride, mannitol, sorbitol, glucose) and other additives, or asan oily injection in solution, suspension or emulsion in a vegetable oilsuch as olive oil, sesame oil, cottonseed oil or corn oil, propyleneglycol or the like.

124. An oral preparation can be produced by shaping the compound of thepresent invention by a commonly known method after addition of anexcipient (e.g., lactose, sucrose, starch), a disintegrant (e.g.,starch, calcium carbonate), a binder (e.g., starch, gum arabic,carboxymethyl cellulose, polyvinylpyrrolidone, hydroxypropyl cellulose),a lubricant (e.g., talc, magnesium stearate, polyethylene glycol 6000)and other additives, and, where necessary, coating the shaped productfor the purpose of taste masking, enteric dissolution or sustainedrelease by a commonly known method. Coating agents for this purposeinclude, for example, hydroxypropylmethyl cellulose, ethyl cellulose,hydroxymethyl cellulose, hydroxypropyl cellulose, polyoxyethyleneglycol, Tween 80, Prulonic F68, cellulose acetate phthalate,hydroxypropylmethyl cellulose phthalate, hydroxymethyl cellulose acetatesuccinate, Eudragit (produced by Rohm Company, Germany; methacrylicacid/acrylic acid copolymer) and dyes (e.g., iron oxide, titaniumdioxide). For an enteric preparation, an intermediate phase may beprovided between the enteric phase and the drug-containing phase for thepurpose of separation of the two phases by a commonly known method.

125. An external preparation can be produced by compounding the compoundof the present invention as a solid, semi-solid or liquid composition bya commonly known method. Such a solid composition is produced by, forexample, powdering the compound of the present invention as such or inmixture with an excipient (e.g., glycol, mannitol, starch,microcrystalline cellulose), a thickening agent (e.g., natural rubber,cellulose derivative, acrylic acid polymer) and other additives. Such aliquid composition is produced by preparing the compound of the presentinvention as an oily or aqueous suspension in almost the same manner aswith the injection. The semi-solid composition is preferably an aqueousor oily gel, or an ointment. All these compositions may contain pHregulators (e.g., carbonic acid, phosphoric acid, citric acid,hydrochloric acid, sodium hydroxide), preservatives (e.g.,paraoxybenzoic acid esters, chlorobutanol, benzalkonium chloride) andother additives.

126. A suppository is produced by preparing the compound of the presentinvention as an oily or aqueous solid, semi-solid or liquid compositionby a commonly known method. Useful oily bases for such compositionsinclude glycerides of higher fatty acids (e.g., cacao fat, uitepsols,producedby Dynamite Nobel Company, Germany), moderate fatty acids (e.g.,MIGLYOL, produced by Dynamite Nobel Company, Germany), and vegetableoils (e.g., sesame oil, soybean oil, cottonseed oil). Aqueous basesinclude, for example, polyethylene glycols and propylene glycol. Basesfor aqueous gels include, for example, natural rubbers, cellulosederivatives, vinyl polymers and acrylic acid polymers.

BEST MODE FOR CARRYING OUT THE INVENTION

127. The present invention is hereinafter described in more detail bymeans of, but is not limited to, the following reference examples,examples, preparation examples and experimental examples.

128.¹H-NMR spectra are determined with tetramethylsilane as the internalstandard, using the GEMINI 200 (200 MHz) spectrometer (produced byVarian, Ltd.), the LAMBDA 300 (300 MHz) spectrometer (produced by JEOL,Ltd.) or the Bruker AM500 (500 MHz) spectrometer (produced by Bruker);all δ values are shown in ppm. Unless otherwise specifically indicated,“%” is by weight. Yield indicates mol/mol %.

129. The symbols used herein have the following definitions:

130. s: singlet

131. d: doublet

132. t: triplet

133. dt: double triplet

134. m: multiplet

135. br: broad

136. The term “at room temperature” indicates the range from about 15 to25° C., but is not to be construed as strictly limitative.

EXAMPLES Reference Example 1 Production of2-amino-5-phenylthiophen-3-carboxylic Acid Ethyl Ester

137. To a mixture of ethyl cyanoacetate (6.1 g, 50 mmol), sulfur (1.61g, 50 mmol), triethylamine (3.5 ml, 25 mmol), and dimethylformamide (10ml), phenylacetaldehyde (50% solution in diethyl phthalate; 12.05 g, 50mmol) was added dropwise with stirring at 45° C. over 20 minutes. Afterstirring at 45° C. for 9 hours, the reaction mixture was concentratedand the obtained residue was extracted with ethyl acetate, washed withsaline and dried (MgSO₄), the solvent was distilled off under reducedpressure. The residue was chromatographed on silica gel andrecrystallized from ether-hexane to yield pale yellow tabular crystals(5.55 g, 45%).

138. mp 124.5-125.5° C. (lit.; 123-124° C.).

139. Elemental analysis for C₁₃H₁₃NO₂S C (%) H (%) N(%)

140. Calculated 63.13; 5.30; 5.66

141. Found: 62.99; 5.05; 5.63

142.¹H-NMR (300 MHz, CDCl₃) δ: 1.37 (3H, t, J=7.1 Hz), 4.30 (2H, d,J=7.1 Hz), 5.97 (2H, br), 7.17-7.46 (6H, m).

143. IR (KBr): 3448, 3320, 1667, 1590, 1549 cm⁻¹.

Reference Example 2 Production of2-amino-4-methyl-5-(4-methoxyphenyl)thiophen-3-carboxylic Acid EthylEster

144. A mixture of 4-methoxyphenylacetone (16.5 g, 0.10 mol), ethylcyanoacetate (12.2 g, 0.10 mol), ammonium acetate (1.55 g, 20 mmol),acetic acid (4.6 ml, 80 mmol), and benzene (20 ml) was heated andrefluxed for 24 hours, while the water being produced was removed usinga Dean-Stark apparatus. After cooling, the reaction mixture wasconcentrated under reduced pressure and the residue was partitionedbetween dichloromethane and aqueous sodium bicarbonate. After theorganic layer was washed with saline and dried (MgSO₄), the solvent wasdistilled off under reduced pressure. To a solution in ethanol (30 ml)of the obtained residue, were added sulfur (3.21 g, 0.10 mol) anddiethylamine (10.4 ml, 0.10 mol), followed by stirring at 50 to 60° C.for 2 hours, successively the reaction mixture was concentrated. Theobtained residue was extracted with ethyl acetate, washed with salineand dried (MgSO₄), the solvent was distilled off under reduced pressure.The residue was chromatographed on silica gel and recrystallized fromether-hexane to yield light yellow tabular crystals (11.5 g, 40%).

145. mp 79-80° C.

146. Elemental analysis for C₁₅H₁₇NO₃S, C (%) H (%) N (%) S (%)

147. Calculated: 61.83; 5.88; 4.81; 11.01

148. Found: 61.81; 5.75; 4.74; 10.82

149.¹H-NMR (200 MHz, CDCl₃) δ: 1.37 (3H, t, J=7.1 Hz), 2.28(3H, s), 3.83(3H, s), 4.31 (2H, q, J=7.1 Hz), 6.05 (2H, brs), 6.91 (2H, d, J=8.8 Hz),7.27 (2H, d, J=8.8 Hz).

150. IR (KBr): 3426, 3328, 1651, 1586, 1550, 1505, 1485 cm⁻¹.

151. FAB-MS m/z: 291 (M⁺).

Reference Example 3

152. Using some acetone derivatives in place of 4-methoxyphenylacetone,the following compound was obtained in the same manner as in ReferenceExample 2.

153. Yield: 40%

154. mp 64-65° C.

Reference Example 4 Production of[3-ethoxycarbonyl-5-(4-methoxyphenyl)-4-methylthiophen-2-yl]aminomethylenemalonicAcid Diethyl Ester

155. To the compound obtained in Reference Example 2 (10 g, 34.3 mmol),ethoxymethylenemalonic acid diethyl ester (7.45 g, 34.5 mmol) was added,followed by stirring at 120° C. for 2 hours. After the reaction mixturewas cooled, the crystal precipitated was collected by filtration via theaddition of ether, again washed with ether, and dried over phosphoruspentaoxide under reduced pressure to yield yellow crystals (14.2 g,90%).

156. mp 122-123° C.

157.¹H-NMR (200 MHz, CDCl₃) δ: 1.32 (3H, t, J=7.1 Hz), 1.38 (3H, t,J=7.2 Hz), 1.41 (3H, t, J=7.2 Hz), 2.34 (3H, s), 3.85 (3H, s), 4.25 (2H,q, J=7.1 Hz), 4.38 (2H, q, J=7.2 Hz), 4.45 (2H, q, J=7.2 Hz), 6.95 (2H,d, J=8.8 Hz), 7.31 (2H, d, J=8.8 Hz), 8.22 (1H, d, J=13.4 Hz), 12.74(1H, d, J=13.1 Hz).

158. IR (KBr): 2984, 1720, 1707, 1688, 1653, 1599, 1518, 1499 cm⁻¹.

Reference Example 5

159. Using the compound obtained in Reference Example 3 as a startingmaterial, the following compound was obtained in the same manner as inReference Example 4.

160. Yield: 92%

161. mp 108-109° C.

Reference Example 6 Production of3-carboxy-5-[(4-methoxyphenyl)-4-methylthiophen-2-yl]aminomethylenemalonicAcid Diethyl Ester

162. To a solution in dioxane (20 ml) of the compound obtained inReference Example 4 (7.0 g, 15.2 mmol) was added, a solution ofpotassium hydroxide (5.0 g, 75.7 mmol) in ethanol (30 ml) with stirringat 60 to 70° C. After stirring at this temperature for 1 hour, thereaction mixture was kept standing at room temperature for 1 hour. Tothe mixture was added 2 N hydrochloric acid (40 ml, 80 mmol) with icecooling, the reaction mixture was concentrated under reduced pressure.The yellow precipitate was collected by filtration, washed with coldwater-ethanol, and dried over phosphorus pentaoxide under reducedpressure to yield yellow powders (6.1 g, 93%).

163. mp 184-187° C.

164.¹H-NMR (200 MHz, DMSO-d₆) δ: 1.24 (3H, t, J=7.1 Hz), 1.28 (3H, t,J=7.2 Hz), 2.30 (3H, s), 3.80 (3H, s), 4.15 (2H, q, J=7.1 Hz), 4.24 (2H,q, J=7.2 Hz), 7.03 (2H, d, J=8.7 Hz), 7.37 (2H, d, J=8.7 Hz), 8.08 (1H,d, J=13.6 Hz), 12.41 (1H, d, J=13.6 Hz).

165. IR (KBr): 3422, 2980, 1719, 1653, 1607, 1551, 1512 cm⁻¹.

Reference Example 7

166. Using the compound obtained in Reference Example 5 as a startingmaterial, the following compound was obtained in the same manner as inReference Example 6.

167. Yield: 98%

168. mp 187-190° C.

Reference Example 8 Production of4-hydroxy-2-(4-methoxyphenyl)-3-methylthieno[2,3-b]pyridine-5-carboxylic Acid Ethyl Ester

169. With stirring at 120° C., small portions of the compound obtainedin Reference Example 6 (6.0 g, 13.8 mmol) was added to polyphoshoricacid ester (PPE) (90 ml). After stirring at the same temperature for 30minutes, the reaction mixture was poured into ice water and extractedwith ethyl acetate. The combined extracts were washed with saline, anddried (MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yieldyellowpowders (3.65 g, 77%). For asample for elemental analysis, the powdersobtained were recrystallized from ethanol to yield yellow crystals.

170. mp 162-163° C.

171. Elemental analysis for C₁₈H₁₇NO₄S C (%) H (%) N (%) S (%)

172. Calculated: 62.96; 4.99; 4.08; 9.34

173. Found: 62.89; 5.04; 4.01; 9.34

174.¹H-NMR (200 MHz, CDCl₃) δ: 1.47 (3H, t, J=7.1 Hz), 2.63 (3H, s),4.87 (3H, s), 4.49 (2H, q, J=7.1 Hz), 6.99 (2H, d, J=8.8 Hz), 7.44 (2H,d, J=8.8 Hz), 8.84 (1H,s), 12.11 (1H, s).

175. IR (KBr): 3434, 2992, 1692, 1601, 1582, 1535, 1504 cm⁻¹.

176. FAB-MS m/z: 344 (MH⁺).

Reference Example 9

177. Using the compound obtained in Reference Example 7 as a startingmaterial, the following compound was obtained in the same manner as inReference Example 8.

178. Yield: 60%

179. mp 155-157° C.

Reference Example 10 Production of4-hydroxy-2-(4-nitrophenyl)-3-methylthieno[ 2,3-b]pyridine-5-carboxylicAcid Ethyl Ester

180. To a solution in concentrated sulfuric acid (10 ml) of the compoundobtained in Reference Example 9 (3.76 g, 12.0 mmol) was added dropwise asolution of sodium nitrate (1.27 g, 15.0 mmol) in concentrated sulfuricacid (5 ml) with ice cooling. After stirring at this temperature for 30minutes, the reaction mixture was poured into ice water and extractedwith chloroform. The combined extracts were washed with saline, anddried (MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield yellowpowders, which was recrystallized from ethanol to yield yellow crystals(1.75 g, 41%).

181. mp 260-261° C.

182. Elemental analysis for C₁₇H₁₄N₂O₅S C (%) H (%) N (%)

183. Calculated: 56.98; 3.94; 7.82

184. Found:56.66; 3.91; 7.86

185.¹H-NMR (200 MHz, CDCl₃) δ: 1.49(3H, t, J=7.1 Hz), 2.70(3H, s), 4.51(2H, q, J=7.1 Hz), 7.70(2H, d, J=8.8 Hz), 8.34(2H, d, J=8.8 Hz),8.89(1H,s), 12.27(1H, s).

186. IR (KBr): 3002, 1692, 1605, 1514, 1350, 1290 cm⁻¹.

187. FAB-MS m/z: 358 (MH⁺).

Reference Example 11 Production of4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-3-methyl-4-oxothieno[2,3-b]pyridine-5-carboxylicAcid Ethyl Ester

188. To a suspension of sodium hydride (60% dispersion in oil; 123 mg,3.08 mmol) in dimethylformamide (3 ml) was added a solution of thecompound obtained in Reference Example 8 (1.0 g, 2.91 mol) indimethylformamide (20 ml) under nitrogen stream with ice cooling. Afterstirring at this temperature for 30 minutes, to the mixture was addeddropwise a solution of 2-methoxybenzyl chloride (0.92 g, 5.87 mmol) indimethylformamide (3 ml). After stirring at room temperature for 23hours and at 70° C. for 2 hours, the reaction mixture was concentrated,and then the obtained residue was partitioned between ethyl acetate andan aqueous solution of ammonium chloride. The water layer was extractedwith ethyl acetate. The combined extracts were washed with saline, anddried (MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield light yellowamorphous powders (0.95 g, 70%). For a sample for elemental analysis,the obtained powders were recrystallized from dichloromethane-ether toyield yellow prismatic crystals.

189. mp 165-167° C.

190. Elemental analysis for C₂₆H₂₅NO₅S 0.5H₂O C (%) H (%) N (%)

191. Calculated: 66.08; 5.55; 2.96

192. Found: 66.33; 5.44; 2.74

193.¹H-NMR (200 MHz, CDCl₃) δ: 1.41(3H, t, J=7.1 Hz), 2.65(3H, s),3.85(3H, s), 3.86(3H, s), 4.39(2H, q, J=7.1 Hz), 5.16(2H, s),6.92-7.00(4H, m), 7.21-7.41(4H, m), 8.41(1H, s).

194. IR (KBr): 2980, 1727, 1684, 1609, 1590, 1497, 1464 cm⁻¹.

Reference Example 12

195. The following compound was produced by the same method as describedin Reference Example 11, using benzyl 2,6-difluorochloride in place of2-methoxybenzyl chloride with the compound obtained in Reference Example10 as the starting material.

196. Yield: 81%

197. mp 215-217° C.

Reference Example 13 Production of3-bromomethyl-4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-5-carboxylic Acid Ethyl Ester

198. A mixture of the compound obtained in Reference Example 11 (0.35 g,0.755 mmol), N-bromosuccinimide (0.135 g, 0.758 mmol),α,α′-azobisisobutyronitrile (13 mg, 0.079 mmol), and carbontetrachloride (5 ml) was heated and refluxed for 2 hours. After cooling,the insoluble substances were filtered off, and then the filtrate wasdiluted with chloroform. After the organic layer was washed with salineand dried (MgSO₄), the solvent was distilled off under reduced pressure.The obtained residue was recrystallized from ethyl acetate to yieldcolorless needles (0.272 g, 66%).

199. mp 200-201° C.

200. Elemental analysis for C₂₆H₂₄NO₅SBr C (%) H (%) N (%)

201. Calculated: 57.57; 4.46; 2.58

202. Found: 57.75; 4.31; 2.31

203.¹H-NMR (200 MHz, CDCl₃) δ: 1.40(3H, t, J=7.1 Hz), 3.86(6H, s),4.40(2H, q, J=7.1 Hz), 5.05(2H, s), 5.16(2H, s), 6.92-7.04(4H, m),7.23-7.28(1H,m), 7.34-7.43(1H,m), 7.57(2H, d, J=8.9 Hz), 8.46(1H, s).

204. IR (KBr): 2980, 1725, 1607, 1588, 1497 cm⁻¹.

Reference Example 14

205. Using the compounds obtained in Reference Examples 12, 22 and 27 asa starting material, respectively, the following compounds (ReferenceExamples 14-1 to 14-3) were obtained in the same manner as in ReferenceExample 13.

Reference Example 14-1

206.

207. Yield: 81%

208. mp 200-202° C.

Reference Example 14-2

209.

210. Yield: 80%

211. Amorphous.

Reference Example 14-3

212.

213. Yield: 79%

214. mp 189-192° C.

Reference Example 15 Production of3-benzylaminomethyl-4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-4-oxothieno[2,3-b]pyridine-5-carboxylic Acid Ethyl Ester Hydrochloride

215. To a solution of the compound obtained in Reference Example 13(0.245 g, 0.452 mmol) in dimethylformamide (5 ml) were addedtriethylamine (0.10 ml, 0.717 mmol) and benzylamine (80 μl, 0.732 mmol)with ice cooling. After stirring at room temperature for 1.5 hours, thereaction mixture was concentrated, and then the obtained residue waspartitioned between ethyl acetate and saturated aqueous sodiumbicarbonate. The water layer was extracted with ethyl acetate. Thecombined organic extracts were dried (MgSO₄), the solvent was distilledoff under reduced pressure. The obtained residue was chromatographed onsilica gel to yield a colorless oil (0.135 g, 53%). To a solution ofthis oil in ethanol (4 ml) was added 10N ethanolic hydrochloric acid(0.2 ml) with ice cooling, followed by stirring at this temperature for10 minutes. The reaction mixture was concentrated, and then the obtainedresidue was crystallized from ethyl acetate-ether to yield thehydrochloride (0.113 g) as white crystals.

216. mp [Hydrochloride] 118-119° C.

217. Elemental analysis for C₃₃H₃₂N₂O₅S HCl 0.9H₂O C (%) H (%) N (%)

218. Calculated: 63.79; 5.64; 4.51

219. Found: 64.03; 5.44; 4.51

220.¹H-NMR (300 MHz, CDCl₃) [Free amine]δ1.40(3H, t, J=7.1 Hz), 2.05(1H,br),3.81(3H, s), 3.86(3H, s), 3.87(2H, s), 3.94(2H, s), 4.40(2H, q,J=7.1 Hz), 5.18(2H, s), 6.80(2H, d, J=8.8 Hz), 6.91-6.99(2H, m),7.20-7.42(9H, m), 8.45(1H, s).

221. IR (KBr) [Hydrochloride]: 3422, 2938, 1719, 1605, 1560,1545, 1502,1460 cm⁻¹.

Reference Example 16 Production of4-methyl-2-[(4-methyl-3-oxo-1-penten-1-yl)amino]-5-phenylthiophen-3-carboxylicAcid Ethyl Ester

222. The compound obtained in Reference Example 3 (10 g),1-methoxy-4-methyl-1-penten-3-one of 85% content (6.9 g),p-toluenesulfonic acid monohydrate (0.219 g), and toluene (100 ml) weremixed and stirred at room temperature for 2.5 hours. After the reactionmixture was diluted with ethyl acetate and washed with a saturatedaqueous solution of sodium hydrogen carbonate, the water layer wasextracted with ethyl acetate. The combined extracts were washed withsaturated saline, the organic layers were dried over anhydrous magnesiumsulfate. The residue was crystallized via the addition of a seedcrystal, after which it was triturated with hexane, collected byfiltration, and washed, to yield the title compound (12.64 g, 92%).

223. mp 104-108° C.

Reference Example 17 Production of4,7-dihydro-7-(2-methoxybenzyl)-2-(4-methoxyphenyl)-3-methyl-4-oxothieno[2,3-b]pyridine-N-benzylpiperazinyl-5-carboxamide

224. To 1-benzylpiperazine (0.77 g, 4.37 mmol) was added dropwise asolution of diisobutyl aluminum hydride (DIBAL) in toluene (1.5N, 2.9ml, 4.37 mmol) with ice cooling. After the addition was completed, thesolution was allowed to warm to room temperature and stirred for further0.5 hours. To this solution was added a solution of the compoundobtained in Reference Example 11 (0.50 g, 1.08 mmol) in toluene (5 ml)at room temperature. After the mixture was further stirred at roomtemperature for 15 hours, methylene chloride (30 ml) was added. Afterwater washing, the solution was dried over sodium sulfate, and then thesolvent was concentrated under reduced pressure to yield a solid (1.03g), which was recrystallized from methylene chloride-n-hexane to yieldthe title compound (0.48 g, 78%).

225. mp 233-235° C.

226. Elemental analysis for C₃₅H₃₅N₃O₄S ½H₂O C (%) H (%) N (t) S (%)

227. Calculated: 69.75; 6.02; 6.97; 5.32.

228. Found: 69.88; 6.06; 6.98; 5.39.

229.¹H-NMR (300 MHz, CDCl₃) δ: 2.45-2.55(4H, m), 2.63(3H, S),3.43-3.49(2H, m), 3.55(2H, s), 3.73-3.82(2H,m), 3.84(6H, s), 5.11(2H,s), 6.89-6.98(4H,m), 7.21-7.40(9H,m), 7.79(1H, s).

Reference Example 18

230. Using the compound obtained in Reference Example 12 as a startingmaterial and N,O-dimethylhydroxy in place of 1-benzylpiperazine, thefollowing compound was obtained in the same manner as in ReferenceExample 17.

231. Yield: 80%

232. mp 223-224° C.

Reference Example 19 Production of4-hydroxy-5-isobutyryl-3-methyl-2-phenylthieno[ 2,3-b]pyridine

233. The compound obtained in Reference Example 16 (50 g) and diphenylether (500 ml) were mixed, and then this mixture was heated and refluxedfor 4 hours, while the ethanol being produced with the progress of thereaction was distilled off. After cooling, the diphenyl ether wasdistilled off under reduced pressure, and then the crude crystalprecipitated was washed with n-hexane to yield the title compound (35.1g, 81%).

234. mp 114-117° C.

Reference Example 20

235. Using the compound obtained in Reference Example 14-1 as startingmaterial and N-methylbenzylamine in place of benzylamine, the followingcompounds (Reference Examples 20-1 to 20-3) were obtained in the samemanner as in Reference Example 15.

Reference Example 20-1

236.

237. Yield: 83%

238. mp 140-144° C.

Reference Example 20-2

239.

240. Yield: 91%

241. mp 145-147° C.

Reference Example 20-3

242.

243. Yield: 78%

244. mp 175-177° C.

Reference Example 21

245. Using the compounds obtained in Reference Example 20 as startingmaterials, the following compounds (Reference Examples 21-1 to 21-3)were obtained in the same manner as in Reference Example 28.

Reference Example 21-1

246.

247. Yield: 79%

248. mp 158-160° C.

Reference Example 21-2

249.

250. Yield: 96%

251. mp 195-196° C.

Reference Example 21-3

252.

253. Yield: 71%

254. mp 144-146° C.

Reference Example 22 Production of7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-3-methyl-4-oxo-2-phenyl-thieno[2,3-b]pyridine

255. The compound obtained in Reference Example 19 (35 g), potassiumcarbonate (18.6 g), and N,N-dimethylformamide (280 ml) were mixed, andthen 2,6-difluorobenzyl bromide (27.9 g) was added, followed by stirringat room temperature for 4 hours. To the reaction mixture was addeddropwise water (560 ml), followed by stirring for 30 minutes, and thenthe mixture was stirred for 1 hour with ice cooling. The crude crystalwas collected by filtration, washed with water, and dried, successivelysuspended in a 1:1 mixed solvent (250 ml) of ethyl acetate anddiisopropyl ether and stirred at 25 to 40° C. for 1 hour, then stirredwith ice cooling for 1 hour, the residual crystal was collected byfiltration and washed with the above mixed solvent (125 ml) to yield thetitle compound (44.6 g, 92%).

256. mp 205-207° C.

Reference Example 23

257. Using the compounds obtained in Reference Example 14-2 andReference Example 24 as starting materials and N-methylbenzylamine inplace of benzylamine, the following compounds (Reference Examples 23-1to 23-2) were obtained in the same manner as in Reference Example 15.

Reference Example 23-1

258.

259. Yield: 83%

260. mp 197-199° C.

Reference Example 23-2

261.

262. Yield: 66%

263. mp 151-152° C.

Reference Example 24 Production of3-bromomethyl-7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-2-(4-nitrophenyl)-4-oxothieno[2,3-b]pyridine

264. After the compound obtained in Reference Example 14-3 (1 g) wasdissolved in methanesulfonic acid (5 ml) with cooling at 10 to 12° C., asolution of sodium nitrate (0.165 g) in methanesulfonic acid (2.5 ml)was added dropwise to this mixture. After being stirred as was for 2hours, the mixture was poured into cold water, and then the crystalprecipitated was collected by filtration, washed with water anddiisopropyl ether, and dried to yield a crude product (1.04 g), whichwas then triturated with ethyl acetate (15 ml), then cooled with ice,filtered, and washed with cold ethyl acetate to yield the title compound(0.647 g, 59.5%).

265. mp 202 to 204° C. (recrystallized from methanol).

Reference Example 25 Production of4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-3-(N-benzyl-N-methylaminomethyl)-4-oxothieno[2,3-b]pyridine-5-(N-isopropyl)carboxamide

266. To a solution of isopropylamine (0.296 g, 5 mmol) in anhydrousmethylene chloride (5 ml) was added dropwise a solution oftrimethylaluminum in hexane (15%, 2.41 ml, 5.0 mmol) at 0° C. After theaddition was completed, the solution was allowed to warm to roomtemperature and further stirred for 1 hour. To this solution was added asolution of the compound obtained in Reference Example 20-2 (0.12 g,0.25 mmol) in anhydrous methylene chloride (3 ml) with ice cooling (0°C.) over 30 minutes. After the mixture was stirred at room temperaturefor further 1 hour, chloroform (50 ml) was added, followed by washingwith water. The combined organic layers were dried with sodium sulfate,and then the solvent was concentrated under reduced pressure to yield asolid, which was recrystallized from chloroformethyl acetate-ethyl etherto yield colorless crystals (0.096 g, 70%).

267. mp 200-202° C.

268.¹H-NMR (500 MHz, CDCl₃)[Free amine]δ: 1.30(6H, d, J=6.7 Hz),2.15(3H, s), 3.66(2H, s), 4.18(2H, s), 4.18-4.31(1H, m), 5.32(2H, s),7.00(2H, t, J=7.26 Hz), 7.13-7.25(5H, m), 7.42(1H, t, J=7.3 Hz),8.02(2H, d, J=8.9 Hz), 8.26(2H, d, J=8.9 Hz), 8.73(1H, s), 10.02(1H, d,J=9.1 Hz).

269. IR (KBr): 2974, 1661, 1597, 1547, 1497, 1346, 1212, 1035 cm⁻¹

270. FAB-Mass m/z 617(MH)⁺

Reference Example 26

271. Using the compound obtained in Reference Example 20-2 as a startingmaterial and N,O-dimethylhydroxyamine, the following compound wasobtained in the same manner as in Reference Example 25.

272. Yield: 96%

273. mp 100-102° C.

Reference Example 27 Production of4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-5-benzoyl-3-methyl-4-oxothieno[2,3-b]pyridine

274. The compound obtained in Reference Example 18 (3.93 g, 7.87 mmol)was dissolved in anhydrous tetrahydrofuran (THF) (300 ml) with slightlywarming. While this solution was kept at 0° C., a solution ofphenylmagnesium bromide in THF (1M, 15.7 ml, 15.7 mmol) was addeddropwise to the mixture over 10 minutes. After the addition wascompleted, the solution was stirred for further 1 hour. The reactionmixture was partitioned between ethyl acetate (300 ml) and water (50ml), and then the water layer was again extracted with ethyl acetate.The combined organic layers were dried with magnesium sulfate, thesolvent was concentrated under reduced pressure to yield a residue,which was chromatographed on silica gel to yield yellow crystals (3.00g, 74%), which was then recrystallized from ethyl acetate-hexane to giveyellow crystals.

275. mp 114-116° C.

276. Elemental analysis for C₂₈H₁₈N₂O₄SF₂ 0.7H₂O C (%) H (%) N (%)

277. Calculated: 63.56; 3.70; 5.29

278. Found 63.83; 3.95; 5.08

279. H-NMR (500 MHz, CDCl₃) δ: 2.68(3H, s), 5.30(2H, s), 7.02(2H, t,J=8.1 Hz), 7.43(3H, t, J=7.2 Hz), 7.52-7.63(3H,m), 7.86(2H, d, J=7.5Hz), 7.99(1H, s), 8.30(2H, d, J=8.7 Hz).

280. IR (KBr): 3422, 3068, 1665, 1615, 1491, 1473, 1346, 853 cm⁻¹.

281. FAB-Mass m/z 517(MH)⁺

Reference Example 28 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-aminophenyl)-5-benzoyl-4-oxothieno[2,3-b]pyridine

282. To a mixture of the compound obtained in Reference Example 23-1(0.30 g, 0.47 mmol) in ethyl alcohol (6 ml) were added water (2 ml), andthen one drop of concentrated hydrochloric acid was added to yield auniform solution. To this solution were added iron powder (0.105 g, 2.0mmol) and concentrated hydrochloric acid (0.39 ml, 4.7 mmol) carefully.After the addition was completed, the mixture was stirred at roomtemperature for 5 hours and filtered through Celite. A small amount ofaqueous ammonia was added, and then the reaction filtrate wasconcentrated under reduced pressure. The obtained residue was pouredinto ice water, neutralized with sodium bicarbonate, and extracted withethyl acetate. After the combined organic layers were washed withsaline, and dried (MgSO₄), the solvent was distilled off under reducedpressure. The obtained residue was chromatographed on silica gel andrecrystallized from isopropyl ether to yield yellow needles (0.24 g,84%).

283. mp 126-128° C.

284. Elemental analysis for C₃₆H₂₉N₃O₂SF₂ ½H₂O C (%) H (%) N (%)

285. Calculated: 68.93; 5.04; 6.70

286. Found: 68.71; 5.18; 6.62

287.¹H-NMR (300 MHz, CDCl₃) δ: 2.13 (3H, s),3.65 (2H, s), 3.87 (2H, brs), 4.14(2H, s), 5.28(2H, s), 6.74(2H, d, J=8.7 Hz), 7.00(2H, t, J=7.8Hz), 7.16-7.24(5H, m), 7.36-7.46(3H, m), 7.53(1H, t, J=7.2 Hz), 7.62(2H,d, J=8.4 Hz), 7.89(2H, d, J=7.2 Hz), 7.94(1H, s).

288. IR (KBr): 3358, 1607, 1495, 1473, 1035 cm⁻¹.

289. FAB-Mass m/z 606(MH)⁺

Reference Example 29 Production of2-(4-aminophenyl)-7-(2,6-difluorobenzyl)-4,7-dihydro-5-isobutyryl-3-(N-benzyl-N-methylaminomethyl)-4-oxothieno[2,3-b]pyridine

290.

291. To a solution of the compound obtained in Reference Example 23-2(0.25g, 0.415 mmol) in methanol (5 ml) were added dropwise iron powder(0.093 g, 1.66 mmol) and concentrated hydrochloric acid (0.8 ml) withice cooling. After the addition was completed, the mixture was stirredat room temperature for 1 hour and filtered through Celite. A smallamount of a saturated aqueous solution of sodium hydrogen carbonate (10ml) was added, followed by extraction with methylene chloride (30 ml×3).The combined extracts were washed with saline, and dried (MgSO₄), thesolvent was distilled off under reduced pressure. The obtained residuewas chromatographed on silica gel to yield light yellow amorphouspowders (0.203 g, 86%).

292.¹H-NMR(300 MHz, CDCl₃)δ: 1.18(6H, d), 2.11(3H, s), 3.65(2H, s),3.85(2H, br s), 4.17(2H, s), 4.18(1H, m), 5.25(2H, s), 6.73(2H, d),6.95(2H, t), 7.10-7.26(5H, m), 7.42(1H, m), 7.58(2H, d), 8.27(1H, s).

Reference Example 30 Production of5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-3-(N-benzyl-N-methylaminomethyl)-2-(4-propionylaminophenyl)-4-oxothieno[2,3-b]pyridine

293. The compound obtained in Reference Example 28 (0.14 g, 0.23 mmol)was dissolved in anhydrous methylene chloride (2 ml), and then to themixture was added triethylamine (0.038 ml) with ice cooling (0° C.).After this solution was stirred for a while, to the mixture was addedpropionyl chloride (0.021 ml, 0.243 mmol). After the addition wascompleted, the solution was further stirred with ice cooling (0° C.) for40 minutes. The reaction mixture was partitioned between methylenechloride (25 ml) and very thinly overlaid water (10 ml). The water layerwas again extracted with methylene chloride (25 ml). The combinedorganic layers were washed with saline, and dried (MgSO₄), the solventwas distilled off under reduced pressure. The solid obtained wasrecrystallized from ethyl acetate-isopropyl ether to yield yellowneedles (0.10 g, 65%).

294. mp 226-228° C.

295. Elemental analysis for C₃₉H₃₃N₃O₃SF₂ 0.7H₂O C (%) H (%) N (%)

296. Calculated: 69.46; 5.14; 6.23

297. Found: 69.60; 5.18; 6.04

298. Thus obtained compound was dissolved in ethyl acetate, and then asaturated solution of hydrogen chloride (HCl) in ether was added inequal to slightly excess amount, and then the crystal precipitated wasrecrystallized from isopropyl ether to yield light yellow needles (0.095g, 61%).

299. mp 218-220° C.

300. Elemental analysis for C₃₉H₃₃N₃O₃SF₂ HCl 3.5H₂O C (%) H (%) N (%)

301. Calculated: 61.53; 5.43; 5.52

302. Found 61.83; 5.33; 5.30

303.¹H-NMR (300 MHz, DMSO-d₆) δ: 1.11(3H, t, J=7.2 Hz),1.93(3H,s),2.35(2H, q,J=7.5 Hz), 3.44(2H, s), 4.00(2H, s), 5.62(2H, s),7.11-7.25(6H, m), 7.43-7.72(10H, m), 7.79(2H, d, J=7.5 Hz), 8.40(1H, s),10.03(1H, s).

304. IR (KBr): 3422, 3068, 1603, 1502, 1473, 1035 cm⁻¹.

305. FAB-Mass m/z 662(MH)⁺

Reference Example 31

306. The following compounds (Reference Examples 31-1 through 31-10)were produced by the same method as described in Reference Example 30,using various acid chlorides, isocyanates, and chlorocarbonic acidesters in place of propionyl chloride, and also using the amines shownin Example 16 and carbonyldiimidazole, with the compounds obtained inReference Examples 28 and 29 as the starting materials. The followingcompounds (Reference Examples 31-11 through 31-16) can also be producedin the same manner.

Reference Example 31-1-1

307.

308. Yield: 68%

309. mp 238-240° C.

Reference Example 31-1-2

310.

311. mp 230-232° C.

Reference Example 31-2-1

312.

313. Yield: 64%

314. mp 201-204° C.

Reference Example 31-2-2

315.

316. mp 207-214° C.

Reference Example 31-3-1

317.

318. Yield: 55%

319. mp 207-210° C.

Reference Example 31-3-2

320.

321. mp 222-226° C.

Reference Example 31-4

322.

323. Yield: 49%

324. mp 185-187° C.

Reference Example 31-5

325.

326. Yield: 79%

327. mp 216-218° C.

Reference Example 31-6

328.

329. Yield: 73%

330. mp 180-183° C.

Reference Example 31-7

331.

332. Yield: 65%

333. mp 245-247° C.

Reference Example 31-8

334.

335. Yield: 65%

Reference Example 31-9

336.

337. Yield: 70%

338. mp 232-234° C.

Reference Example 31-10

339.

340. Yield: 73%

341. mp 192-197° C.

Reference Example 31-11

342.

Reference Example 31-12

343.

Reference Example 31-13

344.

Reference Example 31-14

345.

Reference Example 31-15

346.

Reference Example 31-16

347.

Reference Example 32 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihyrdro-7-(2,6-difluorobenzyl)-2-(4-nitrophenyl)-5-benzoyl-4-oxothieno[2,3-b]pyridine

348. The compound obtained in Reference Example 26 (1.91 g, 3.09 mmol)was dissolved in anhydrous tetrahydrofuran (THF) (30 ml) with warming,and then to the mixture was added dropwise a solution of phenylmagnesiumbromide in THF (1M, 6.18 ml, 6.2 mmol) with ice cooling (0° C.) over 10minutes. After stirring under ice cooling conditions for 1 hour, thereaction mixture was partitioned between ethyl acetate (100 ml) andaqueous hydrochloric acid (0.5N, 100 ml), and then the organic layer wasagain washed with saturated saline (100 ml). After the organic layer wasdried (MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield yellowcrystals (1.00 g, 51%), which was then recrystallized from isopropylether to yield yellow needles.

349. mp 197-199° C.

350. Elemental analysis for C₃₆H₂₇N₃O₄SF₂ 0.7H₂O C (%) H (%) N (%)

351. Calculated: 66.70; 4.42; 6.48

352. Found: 66.59; 4.48; 6.42

353.¹H-NMR (300 MHz, CDCl₃) δ: 2.17(3H, s), 3.61(2H, s), 4.16(2H, s),5.30(2H, s), 7.03(2H, t, J=8.1 Hz), 7.19-7.25(5H, m), 7.40-7.47(3H, m),7.56(1H, t, J=7.5 Hz), 7.88(2H, d, J=6.9 Hz), 7.96(1H, s), 8.10(2H, d,J=8.7 Hz), 8.28(2H, d, J=8.7 Hz).

354. IR (KBr): 3430,1663, 1611, 1518, 1473, 1348, 853 cm⁻¹.

355. FAB-Mass m/z 636(MH)⁺

Reference Example 33 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihyrdro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-(2-bromoisobutyryl)-4-oxothieno[2,3-b]pyridine

356. The compound obtained in Reference Example 31-10 (0.48 g, 0.75mmol) was dissolved in dichloromethane (15 ml). To the solution wasadded dropwise 47% hydrobromic acid (0.35 ml, 3.0 mmol) at roomtemperature for 1 minute, followed by further stirring for 10 minutes.To this reaction mixture was added dropwise bromine (39 μl, 0.75 mmol),followed by further stirring at room temperature for 24 hours. Thereaction mixture was partitioned between dichloromethane (30 ml) andaqueous sodium bicarbonate (saturated, 60 ml), and then the organiclayer was again washed with saturated saline (100 ml). After the organiclayer was dried (MgSO₄), the solvent was distilled off under reducedpressure to yield brown amorphous crystals (0.53 g).

Reference Example 34 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihyrdro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-methacryloyl-4-oxothieno[2,3-b]pyridine

357. The compound obtained in Reference Example 33 (0.52 g, 0.72 mmol)was dissolved in dimethylformamide (DMF) (30 ml), and then to thissolution was added potassium acetate (2.0 g, 20 mmol) at roomtemperature, followed by stirring at 100° C. for 20 hours. Thereactionmixturewas partitioned between ethyl acetate (30 ml) and water(30 ml), and then the water layer was again washed with ethyl acetate(10 ml). The combined organic layers were again washed with saturatedsaline (30 ml). After the organic layer was dried (MgSO₄), the solventwas distilled off under reduced pressure to yield a brown oil (0.56 g).

Example 1 Example 1-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridine

358.

359. To a solution of the compound obtained in Reference Example 29(2.57 g, 4.5 mmol) in dichloromethane (50 ml) were addedcyclopropanecarboxylic acid (861 mg, 10 mmol) and diisopropylethylamine(2.59 g, 20 mmol), and then benzotriazol-1-yl-oxy tripyrrolidinophosphonium hexafluorophosphoric acid (PyBop) (5.40 g, 10 mmol) wasadded to it in a small portion with ice cooling. After stirring at roomtemperature for 1 day, the reaction mixture was poured into a 0.1Naqueous solution of potassium hydroxide and extracted with chloroform.The combined extracts were washed with saline, and dried (Na₂SO₄), thesolvent was distilled off under reduced pressure. The obtained residuewas chromatographed on silica gel and recrystallized from ethyl acetateto yield white powdery crystals (2.15 g, 79%).

360.¹H-NMR (300 MHz, CDCl₃) δ: 0.85-0.90(2H,m), 1.10-1.15(2H,m),1.18(6H, d), 1.53-1.59(1H,m), 2.09(3H, s), 4.12-4.20(1H, m), 4.16(2H,s), 5.25(2H, s), 6.99(2H, t), 7.10-7.25(6H, m), 7.35-7.45(1H, m),7.61(2H, d), 7.67(1H, br s), 7.76(2H, d), 8.27(1H, s).

361. FAB-Mass m/z 608(MH)⁺

Example 1-2 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridineHydrochloride

362.

363. The white powdery crystals obtained in Example 1-1 were dissolvedin ethanol, and then a solution of hydrogen chloride in ethyl ether wasadded to it in a small excess amount, followed by recrystallization fromethanol-ethyl ether, to yield white powdery crystals (2.20 g, 72%).

364. mp 254-256° C.

365. Elemental analysis for C₃₇H₃₅N₃O₃SF₂ HCl C (%) H (%) N (%)

366. Calculated 65.72; 5.37; 6.21

367. Found: 65.61; 5.38; 6.25

368. IR (KBr): 2950, 1673, 1595, 1502, 1473, 1408, 1313, 1183 cm⁻¹.

Example 2 Production of4,7-dihydro-2-[4-(3-acetoxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

369.

370. To a solution of the compound obtained in Reference Example 29(1.14 g, 2.0 mmol) in dichloromethane (30 ml) were added dropwisetriethylamine (500 mg, 5 mmol) and 3-acetoxyisobutyryl chloride (0.5 g,3 mmol) with ice cooling over 1 minute, followed by further stirring atroom temperature for 1 hour. The reaction mixture was partitionedbetween a saturated aqueous solution of sodium hydrogen carbonate (30ml) and dichloromethane (30 ml), and then the water layer was againextracted with dichloromethane (10 ml). The combined extracts werewashed with saline, and dried (Na₂SO₄), the solvent was distilled offunder reduced pressure. The obtained residue was chromatographed onsilica gel to yield brown crystals (1.4 g).

371.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.30(3H,d), 2.08(3H,s),2.12(3H,s), 2.73-2.84(1H,m), 3.65(2H,s), 4.11-4.36(3H,m), 4.17(2H,s),5.27(2H,s), 7.00(2H,t), 7.13-7.22(5H,m), 7.36-7.46(1H,m), 7.52(1H,br s),7.62-7.82(4H,q), 8.29(1H, s).

Example 3 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridine

372.

373. The compound obtained in Example 2 (1.40 g) was dissolved inmethanol (100 ml), and then to the solution was added potassiumcarbonate (2.0 g, 15 mmol), followed by stirring at room temperature for15 minutes. The reaction mixture was partitioned between ethyl acetate(200 ml) and distilled water (300 ml), and then the water layer wasextracted with ethyl acetate (100 ml). The combined extracts were washedwith saline, and dried (Na₂SO₄), the solvent was distilled off underreduced pressure. The obtained residue was chromatographed on silica geland recrystallized from ether to yield white crystals (0.95 g, 72%).

374. mp 161-162° C.

375. Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 0.5H₂O C (%) H (%) N (%)

376. Calculated: 66.65; 5.74; 6.30

377. Found: 66.70; 5.52; 6.40

378.¹H-NMR (300 MHz, CDCl₃) δ: 1.17(6H,d), 1.18(3H,d), 2.06(3H,s),2.67-2.78(1H,m), 3.61(2H,s), 3.57-3.84(2H,m), 3.99-4.08(1H,m),4.15(2H,s), 5.52(2H,s), 7.08(2H,t), 7.15(5H,s), 7.44-7.54(1H,m),7.58(2H,d), 7.69(2H,d), 8.46(1H, s).

379. FAB-Mass m/z 658(MH)⁺

Example 4

380. Using the compound obtained in Reference Example 29, the followingcompounds (Examples 4-1 to 4-3) were obtained in the same manner as inExample 1.

Example 4-1

381.

382. Yield: 67%

383. mp 201-203° C.

Example 4-2

384.

385. Yield: 58%

386. mp 135-137° C.

Example 4-3

387.

388. Yield: 75%

389. mp 205-207° C.

Example 5 Example 5-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-[4-(2-hydroxyacetylamino)phenyl]-5-isobutyryl-4-oxothieno[2,3-b]pyridine

390.

391. The compound obtained in Example 4-1 (0.137 g) was dissolved indichloromethane (1.0 ml), and then to the solution was added dropwisetrifluoroacetic acid (1.0 ml) with ice cooling. After stirring under icecooling conditions for 1 hour, the reaction mixture was concentrated todryness under reduced pressure, and then the obtained residue waspartitioned between chloroform (50 ml) and saturated aqueous sodiumbicarbonate (50 ml), and then the water layerwas again extracted withchloroform (10 ml). The combined organic layers were washed withsaturated saline (30 ml). After the organic layer was dried (MgSO₄), thesolvent was distilled off under reduced pressure, and then the obtainedresidue was chromatographed on silica gel and recrystallized fromethanol-ethyl acetate-ethyl ether to yield white crystalline powders(0.12 g, 95%).

Example 5-2

392. Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-[4-(2-hydroxyacetylamino)phenyl]-5-isobutyryl-4-oxothieno[2,3-b]pyridineHydrochloride

393. The white crystalline powders obtained in Example 5-1 weredissolved in dichloromethane (1.0 ml), and then to this solution wasadded an ethanolic solution containing 10N hydrogen chloride (1.0 ml)with ice cooling. The solvent was distilled off under reduced pressure,and then the residue was recrystallized from ethanol-ethyl acetate toyield white crystalline powders (0.12 g, 84%).

394. mp 197-199° C.

395. Elemental analysis for C₃₅H₃₃N₃O₄SF₂ HCl 2.5H₂O C (%) H (%) N (%)

396. Calculated: 59.11; 5.53; 5.91

397. Found: 59.06; 5.25; 5.93

398.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H, d), 2.10(3H, s), 3.63(2H,s),3.72(1H,br), 4.09-4.17(1H,m), 4.16(2H,s), 4.24(2H,s), 5.24(2H,s),6.99(2H,t), 7.18-7.23(5H,m), 7.35-7.45(1H,m), 7.63(2H,d), 7.73(2H,d),8.25(1H,s), 8.59(1H,s).

Example 6

399. Using the compounds obtained in Example 4-2 and Example 4-3, thefollowing compounds (Examples 6-1 to 6-2) were obtained in the samemanner as in Example 5.

Example 6-1

400.

401. Yield: 76%

402. mp 192-194° C.

Example 6-2

403.

404. Yield: 81%

405. mp 193-195° C.

Example 7 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-(2-hydroxy-2-methylpropionyl)-4-oxothieno[2,3-b]pyridine

406.

407. The brown amorphous crystal (0.13 g) obtained by repeating themethod described in Reference Example 33 using the compound obtained inReference Example 31-10 was hydrolyzed with potassium acetate by thesame method as described in Reference Example 34. The solid obtained waschromatographed on silica gel to remove the compound obtained inReference Example 34, and the byproduct was recrystallized from methanolto yield colorless crystalline powders (0.03 g).

408. mp 170-172° C.

409. Elemental analysis for C₃₇H₃₇N₃O₄SF₂ ⅙H₂O C (%) H (%) N (%)

410. Calculated: 67.26; 5.69; 6.36

411. Found: 67.18; 5.74; 6.46

412.¹H-NMR (300 MHz, CDCl₃) δ: 1.28(6H,d), 1.49(6H,s), 2.06(3H,s),2.51-2.60(1H,m), 3.61(2H,s), 4.13(2H,s), 5.31(2H,s), 7.01(2H,t),7.12-7.21(5H,m), 7.37-7.47(2H,m), 7.64(2H,d), 7.74(2H,d), 8.04(1H,s),8.35(1H,s).

413. IR (KBr): 3466,1669, 1499, 1313, 1075 cm⁻¹.

Example 8 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-7-(2,6-difluorobenzyl)-2-(4-isobutyrylaminophenyl)-5-(3-hydroxy-2-methylpropionyl)-4-oxothieno[2,3-b]pyridine

414.

415. The compound obtained in Reference Example 34 (0.56 g) wasdissolved in trifluoroacetic acid (10 ml) and stirred at roomtemperature for 1 hour. The reaction mixture was concentrated to drynessunder reduced pressure, and the obtained residue was partitioned betweendichloromethane (30 ml) and saturated aqueous sodium bicarbonate (60ml), and then the water layer was again extracted with dichloromethane(10 ml). The combined organic layers were washed with saturated saline(30 ml). After the organic layer was dried (MgSO₄), the solvent wasdistilled off under reduced pressure to yield brown amorphous crystals(0.42 g), which was chromatographed on silica gel to yield yellowcrystals (0.07g, overall recovery rate as calculated from the compoundobtained in Reference Example 31-10 12%).

416.¹H-NMR (300 MHz, CDCl₃) δ: 1.15(3H,d), 1.26(6H, d), 2.07(3H, s),2.53-2.62(1H, m), 3.58(2H, d), 3.66(2H, d), 3.82(1H, dd), 3.93(1H, dd),4.03(1H, d), 4.22(1H, d), 4.27-4.37(1H, m), 5.26(2H, s), 6.99(2H, t),7.13-7.21(5H, m), 7.36-7.46(1H,m), 7.63(2H, d), 7.67(2H, d), 7.79(1H,s), 8.30(1H, s).

Example 9 Production of(R)-4,7-dihydro-2-[4-(3-t-butoxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

417.

418. To a solution of the compound obtained in Reference Example 29(1.14 g, 2.0 mmol) in dichloromethane (20 ml) were addeddiisopropylethylamine (520 mg, 4 mmol) and(2R)-3-t-butoxy-2-methylpropanoic acid (0.43 g, 3 mmol), followed bystirring with ice cooling. To this solution was addedbenzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate(BOP reagent) (1.33 g, 3 mmol). After stirring with ice cooling for 1hour, the solution was further stirred at room temperature for4 hours.The reaction mixture was concentrated to dryness under reduced pressure,and then the obtained residue was partitioned between water (50 ml) andchloroform (50 ml). The water layer was again extracted with chloroform(10 ml). The combined extracts were washed with saline, and dried(MgSO₄), the solvent was distilled off under reduced pressure. Theobtained residue was chromatographed on silica gel to yield yellowpowders (1.11 g, 78%).

419.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.25(3H,d), 1.30(9H,s),2.62(3H,s), 2.63-2.71(1H,m), 3.51(1H,t like), 3.61(1H,dd), 3.64(2H,s),4.18(2H,s), 4.18(1H,quint), 5.27(2H,s), 7.00(2H,t), 7.14-7.22(5H,m),7.36-7.46(1H,m), 7.60(2H,d), 7.76(2H,d), 8.28(1H, s), 9.00(1H,s).

Example 10 Example 10-1 Production of(R)-4,7-dihydro-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

420.

421. The compound obtained in Example 9 (1.11 g) was dissolved intrifluoroacetic acid (5 ml) under ice cooling conditions, and then afterstirring for 1 hour, the solution was allowed to warm to roomtemperature and stirred for further 12 hours. The reaction mixture wasconcentrated to dryness under reduced pressure, and then the residue wasdissolved in methanol (20 ml) and again cooled with ice, and then a 5Nsolution of sodium hydroxide was added to reach pH 9.0. The reactionmixture was stirred for 30 minutes and further stirred at roomtemperature for 30 minutes. The reaction mixture was concentrated todryness under reduced pressure at low temperature, and then the obtainedresidue was chromatographed on silica gel. The light yellow amorphouscrystals obtained (0.83 g, 82%) were recrystallized from ether to yieldlight yellow powdery crystals (0.69 g).

422. mp 161-162° C.

423. Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 0.5H₂O C (%) H (%) N (%)

424. Calculated: 66.65; 5.74; 6.30

425. Found: 66.47; 5.73; 6.10

426.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.28(3H,d), 2.11(3H,s),2.71(1H,m), 3.65(2H,s), 3.83-3.85(2H,m), 4.16(1H,quint), 4.18(2H,s),5.27(2H,s), 7.00(2H,t), 7.14-7.23(5H,m), 7.36-7.44(1H,m), 7.64(2H,d),7.77(2H,d), 8.09(1H, s), 8.28(1H,s).

427. FAB-Mass m/z 658(MH)⁺

Example 10-2 Production of(R)-4,7-dihydro-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridineHydrochloride

428.

429. The title compound was obtained in the same manner as in Example 1.

430. mp 214-216° C.

Example 11 Example 11-1 Production of4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

431.

432. To a solution of the compound obtained in Reference Example 29(1.14 g, 2.0 mmol) in dichloromethane (30 ml) were addeddiisopropylethylamine (1.04 g, 8 mmol) and 2-hydroxyisobutanoic acid(0.416 g, 4 mmol), followed by stirring under ice cooling conditions. Tothis solution was added benzotriazol-1-yloxytrisdimethylaminophosphoniumhexafluorophosphate (BOP reagent) (1.76 g, 4 mmol). After stirring underice cooling conditions for 1 hour, the solution was further stirred atroom temperature for 4 days. The reaction mixture was concentrated todryness under reduced pressure, and then the obtained residue waspartitioned between water (50 ml) and chloroform (50 ml). The waterlayer was again extracted with chloroform (10 ml). The combined extractswere washed with saline, and dried (MgSO₄), the solvent was distilledoff under reduced pressure. The obtained residue was chromatographed onsilica gel and recrystallized from ether to yield yellow powderycrystald (0.56 g, 43%).

433. mp 178-180° C.

434. Elemental analysis for C₃₇H₃₇N₃O₄SF₂ 0.5H₂O C (%) H (%) N (%)

435. Calculated: 66.65; 5.74; 6.30

436. Found: 66.54; 5.49; 6.36

437.

438.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.58(6H,s), 2.10(3H,s),3.05(1H,brs), 3.64(2H,s), 4.16(2H,s), 4.12-4.22(1H,m), 5.23(2H,s),6.99(2H,t), 7.05-7.25(5H,m), 7.34-7.44(1H,m), 7.64(2H,d), 7.75(2H,d),8.25(1H, s), 8.86(1H, s).

Example 11-2

439. Production of4,7-dihydro-2-(4-(2-hydroxy-2-methylpropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridineHydrochloride

440. The title compound was obtained in the same manner as in Example 1.

441. mp 213-215° C.

Example 12 Example 12-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine

442.

443. To a solution of the compound obtained in Reference Example 29(0.57 9, 1.0 mmol) in dichloromethane (10 ml) were addeddiisopropylethylamine (0.52 g, 4 mmol) and2-hydroxycyclopropanecarboxylic acid (0.204 g, 2 mmol), followed bystirring under ice cooling conditions. To this solution was addedbenzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate(BOP reagent) (1.76 g, 4 mmol). After stirring under ice coolingconditions for 1 hour, the solution was further stirred at roomtemperature for 4 days. The reaction mixture was concentrated to drynessunder reduced pressure, and then the obtained residue was partitionedbetween water (50 ml) and chloroform (50 ml). The water layer was againextracted with chloroform (10 ml). The combined extracts were washedwith saline, and dried (MgSO₄), the solvent was distilled off underreduced pressure. The obtained residue was chromatographed on silica geland recrystallized from ether to yield yellow powdery crystals (0.27 g,41%).

444.¹H-NMR (300 MHz, CDCl₃) δ: 1.16-1.20(2H,m), 1.18(6H,d),1.48-1.51(2H,m), 2.09(3H,s), 3.64(2H,s), 3.95(1H,br s), 4.14(2H,s),4.12-4.19(1H,m), 5.20(2H,s), 6.99(2H,t), 7.10-7.25(5H,m),7.34-7.46(1H,m), 7.57(2H,d), 7.70(2H,d), 8.21(1H, s), 8.82(1H, s).

Example 12-2 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine Hydrochloride

445.

446. The title compound, white powdery crystals (0.12 g) was obtained inthe same manner as in Example 1.

447. Solvent for recrystallization: ethanol-ether

448. mp 220-222° C.

449. Elemental analysis for C₃₇H₃₅N₃O₄SF₂ HCl 0.5H₂O C (%) H (%) N (%)

450. Calculated: 63.37; 5.32; 5.99

451. Found: 63.35; 5.24; 5.82

Example 13 Example 13-1 Production of4,7-dihydro-2-[4-(3-hydroxy-3-methylbutyrylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

452.

453. To a solution of the compound obtained in Reference Example 29(1.14 g, 2.0 mmol) in dichloromethane (10 ml) were addeddiisopropylethylamine (1.04 g, 8 mmol) and 3-hydroxy-3-methylbutanoicacid (0.47 g, 4 mmol), followed by stirring under ice coolingconditions. To this solution was addedbenzotriazol-1-yloxytrisdimethylaminophosphonium hexafluorophosphate(BOP reagent) (1.76 g, 4 mmol). After stirring under ice coolingconditions for 1 hour, the solution was further stirred at roomtemperature for 4 days. The reaction mixture was concentrated to drynessunder reduced pressure, and then the obtained residue was partitionedbetween water (50 ml) and chloroform (50 ml). The water layer was againextracted with chloroform (10 ml). The combined extracts were washedwith saline, and dried (MgSO₄), the solvent was distilled off underreduced pressure. The obtained residue was chromatographed on silica geland recrystallized from ether to yield yellow powdery crystals (0.50 g,37%).

454.¹H-NMR (300 MHz, CDCl₃) δ: 1.17(6H,d), 1.39(6H,s), 2.11(3H,s),2.58(2H,s), 3.12(1H, br s), 3.65(2H,s), 4.12-4.19(1H,m), 4.18(2H,s),5.27(2H,s), 7.00(2H,t), 7.10-7.23(5H,m), 7.32-7.44(1H,m), 7.61(2H,d),7.79(2H,d), 8.24(1H, s), 8.28(1H, s).

Example 13-2 Production of4,7-dihydro-2-[4-(3-hydroxy-3-methylbutyrylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridineHydrochloride

455.

456. The title compound, white powdery crystals (0.42 g) were obtainedin the same manner as in Example 1.

457. Solvent for recrystallization: ethanol

458. mp 216-218° C.

459. Elemental analysis for C₃₈H₃₉N₃O₄SF₂ HCl H₂O C (%) H (%) N (%)

460. Calculated: 62.84; 5.83; 5.79

461. Found: 62.70; 5.75; 5.82

Example 14

462. The following compounds (Examples 14-1 through 14-10) were producedby the same method as described in Examples 9, 11, 12, and 13, using thecompound obtained in Reference Example 29, and also using variouscarboxylic acid compounds in place of, for example, (2R)-3-t-butoxy-2-methylpropanoic acid, which was used in Example 9.

Example 14-1

463.

464. Yield: 46%

465. Amorphous.

466.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H,d), 1.25(3H,d), 1.30(9H,s),2.11(3H,s), 2.60(1H,br), 3.51(1H, t like), 3.59-3.65(1H,m), 3.65(2H,s),4.17(1H,q), 4.18(2H,s), 5.27(2H,s), 7.00(2H,t), 7.12-7.22(5H,m),7.38-7.44(1H,m), 7.60(2H,d), 7.75(2H,d), 8.28(1H, s), 9.00(1H, s).

Example 14-2

467.

468. Yield: 47%

469. mp 145-150° C.

Example 14-3

470.

471. Yield: 99%

472. mp 96-98° C.

Example 14-4

473.

474. Yield: 57%

475. mp 92-94° C.

Example 14-5

476.

477. Yield: 71%

478. mp 57-62° C.

Example 14-6

479.

480. Yield: 92%

481. mp 79-81° C.

Example 14-7

482.

483. Yield: 97%

484. mp 91-96° C.

Example 14-8

485.

486. Yield: 91%

487. mp 75-80° C.

Example 14-9

488.

489. Yield: 87%

490. mp 83-88° C.

Example 14-10

491.

492. Yield: 50%

493. mp 130-135° C.

Example 15 Using the compound obtained in Example 14, the followingcompounds (Examples 15-1 to 15-6) in the same manner as in Example 10.Example 15-1

494.

495. Yield: 69%

496. mp 161-162° C.

Example 15-2

497.

498. Yield: 94%

499. mp 153-155° C.

Example 15-3

500.

501. Yield: 62%

502. mp 224-226° C.

Example 15-4

503.

504. Yield: 56%

505. mp 230-232° C.

Example 15-5

506.

507. Yield: 90%

508. mp 92-94° C.

Example 15-6

509.

510. Yield: 34%

511. mp 107-112° C.

Example 16 Example 16-1 Production of3-(N-benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-N′-methoxyureidophenyl)-4-oxothieno[2,3-b]pyridine

512.

513. To a solution of the compound obtained in Reference Example 29(0.40 g, 0.7 mmol) in dichloromethane (15 ml) were added triethylamine(0.20 ml, 1.43 mmol) and N,N′-carbonyldiimidazole (0.228 g, 1.41 mmol),followed by stirring under ice cooling conditions. After the solutionwas allowed to warm to room temperature and further stirred for 2.5days, to the mixture were added O-methylhydroxylamine hydrochloride(0.59 g, 7.06 mmol) and triethylamine (0.98 ml, 7.03 mmol) with icecooling. While the solution was allowed to return to room temperature,it was stirred for 1.5 hours. After dilution with water (50 ml), thereaction mixture was extracted with chloroform (50 ml). The water layerwas again extracted with chloroform (10 ml). The combined extracts werewashed with saline, and dried (MgSO₄), the solvent was distilled offunder reduced pressure. The obtained residue was chromatographed onsilica gel to yield yellow amorphous powders (0.349 g, 77%).

514.¹H-NMR (300 MHz, CDCl₃) δ: 1.19(6H,d), 2.17(3H,s), 3.72(2H,brs),3.83(3H,s), 4.10-4.20(1H,m), 4.22(2H,brs), 5.28(2H,s), 7.00(2H,t),7.15-7.24(5H,m), 7.37-7.46(2H,m), 7.59(2H,d), 7.75(2H,d), 7.83(1H, br),8.31(1H,s).

Example 16-2 Production of4,7-dihydro-2-(4-N′-methoxyureidophenyl)-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridineHydrochloride

515.

516. The title compound, white amorphous powders (0.335 g) were obtainedin the same manner as in Example 1.

517. Solvent for recrystallization: ethyl acetate-ether

518. mp 225-230° C.

519. Elemental analysis for C₃₅H₃₄N₄O₄SF₂ HCl 0.5H₂O C (%) H (%) N (%)

520. Calculated: 60.91; 5.26; 8.12

521. Found: 60.80; 5.07; 8.17

Example 17 Example 17-1 Production of(R)-4,7-dihydro-2-[4-(2,3-dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

522.

523. To a 2N aqueous solution of potassium hydroxide was added(R)-(+)-2,2-dimethyl-1,3-dioxolan-4-carboxylic acid methyl ester (1.00g, 6.24 mmol), followed by stirring under ice cooling conditions for 3hours. After neutralization with hydrochloric acid, the solution wassalted out and extracted with ethyl acetate (50 ml) to yield(R)-(+)-2,2-dimethyl-1,3-dioxolan-4-carboxylic acid as colorlessamorphous powders (0.72 g). To a solution of the compound obtained inReference Example 29 (0.572 g, 1.0 mmol) in dichloromethane (5 ml) wereadded diisopropylethylamine (1.04 g, 8 mmol) and the(R)-(+)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid obtained (0.292 g,2 mmol), followed by stirring under ice cooling conditions. To thissolution was added benzotriazol-1-yloxytrisdimethylaminophosphoniumhexafluorophosphate (BOP reagent) (0.885 g, 2 mmol). After stirringunder ice cooling conditions for 1 hour, the solution was furtherstirred at room temperature for 4 days. The reaction mixture wasconcentrated to dryness under reduced pressure, and then the obtainedresidue was partitioned between water (50 ml) and chloroform (50 ml).The water layer was again extracted with chloroform (10 ml). Thecombined extracts were washed with saline, and dried (MgSO₄), thesolvent was distilled off under reduced pressure. The obtained residuewas chromatographed on silica gel to yield colorless amorphous powders(0.5 g), which was then dissolved in trifluoroacetic acid (10 ml) andstirred at room temperature for 16 hours. The reaction mixture wasconcentrated to dryness under reduced pressure and treated with alkali,after which it was partitioned between chloroform (50 ml) and water (50ml), and then the water layer was again extracted with chloroform (10ml). The combined extracts were washed with saline, and dried (MgSO₄),the solvent was distilled off under reduced pressure. The obtainedresidue was chromatographed on silica gel to yield 0.5 g of colorlessamorphous powders (76%).

524.¹H-NMR (300 MHz, DMSO-d₆) δ: 1.09(6H,d), 1.95(3H,s), 3.51(2H,br),3.55-3.72(2H,m), 4.01-4.08(4H,m), 4.84(1H,t), 5.63(2H,brs), 5.80(1H,d),7.15-7.24(7H,m), 7.49-7.57(1H,m), 7.66(2H,d), 7.84(2H,d), 8.53(1H,s),9.82(1H,s).

Example 17-2 Production of(R)-4,7-dihydro-2-[4-(2,3-dihydroxypropionylamino)phenyl]-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridineHydrochloride

525.

526. The title compound, white powdery crystals (0.43 g) were obtainedin the same manner as in Example 1.

527. Solvent for recrystallization: ether-ethanol

528. mp 146-148° C.

Example 18 Production of4,7-dihydro-2-(4-N′-hydroxy-N′-methylureidophenyl)-7-(2,6-difluorobenzyl)-3-(N-benzyl-N-methylaminomethyl)-5-isobutyryl-4-oxothieno[2,3-b]pyridine

529.

530. To a solution of the compound obtained in Reference Example 29 (1.0g, 1.75 mmol) in dichloromethane (40 ml) were added and triethylamine(0.49 ml, 3.50 mmol) and N,N′-carbonyldiimidazole (0.567 mg, 3.50 mmol),followed by stirring under ice cooling conditions. After the solutionwas allowed to warm to room temperature and further stirred for 17hours, to the mixture was added N-methylhydroxylammonium chloride (730mg, 8.75 mmol) and triehylamine (1.23 ml, 8.75 mmol) with ice cooling.While the solution was allowed to warm to room temperature, it wasstirred for 5 hours. The reaction mixture was partitioned betweenchloroform (300 ml) and aqueous sodium bicarbonate (saturated, 200 ml),and then the organic layer was again washed with saline (200 ml). Afterthe organic layer was dried (MgSO₄), the solvent was distilled off underreduced pressure. The residue obtained was chromatographed on silica gelto yield pale yellow amorphous powder (0.38 g, 34%).

531.¹H-NMR (300 MHz, CDCl₃) δ: 1.18(6H, d), 2.15(3H, s), 3.26(3H, s),3.71(2H, s), 4.19(2H, s), 5.25(2H, s), 7.00(2H, t), 7.22-7.26(5H, m),7.41(1H, m), 7.49(2H,d), 7.60(2H,d), 8.06(1H, s), 8.26(1H, s).

532. Elemental analysis for C₃₅H₃₄N₄O₄SF₂ 0.8H₂O C (%) H (%) N (t)

533. Calculated 63.78; 5.44; 8.50

534. Found: 63.73; 5.24; 8.41

Example 19 Production of2-[4-[(1-acetoxycyclopropyl)carbonylaminolphenyl]-3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-4-oxothieno[2,3-b]pyridine

535.

536. To a solution of the compound obtained in Reference Example 28(0.42 g, 0.7 mmol) and triethylamine (0.08 g, 0.8 mmol) indichloromethane (10 ml) was added 1-acetoxycyclopropanecarbonyl chloride(0.13 g, 0.8 mmol) with ice-cooling. The mixture was stirred at 0° C.for 30 minutes and at room temperature for 30 minutes. The reactionmixture was diluted with chloroform (20 ml) and the solution was washedwith aqueous sodium bicarbonate (saturated, 20 ml), and brine (20 ml).After the organic layer was dried (Na₂SO₄), the solvent was distilledoff under reduced pressure. The residue obtained was chromatographed onsilica gel to yield pale yellow amorphous powders (0.34 g, 66%).

537.¹H-NMR (300 MHz, CDCl₃) δ: 1.22(2H, dd), 1.68(2H, dd), 2.12(3H, s),2.21(3H, s), 3.64(2H, s), 4.13(2H, s), 5.28(2H, s), 7.01(2H, t),7.17-7.23(5H, m), 7.37-7.44(3H, m), 7.51-7.56(1H, m), 7.60(2H, d),7.81(2H, d), 7.87-7.90(3H, m) , 7.94(1H, S).

538. FAB-Mass m/z 732 (MH)⁺

Example 20

539. Using the compound obtained in Reference Example 28, the followingcompound Examples 20-1 was obtained in the same manner as in Example 19

Example 20-1

540.

541. Yield: 80%

542.¹H-NMR (300 MHz, CDCl₃) δ: 1.77(6H, s), 2.12(3H, s), 2.17(3H, s),3.63(2H, s), 4.13(2H, s), 5.29(2H, s), 7.01(2H, t), 7.15-7.23(5H, m),7.38-7.45(3H, m), 7.51-7.56(1H, m), 7.64(2H, d), 7.85(2H, d),7.88-7.90(3H, m), 7.94(1H, s).

543. FAB-Mass m/z 734(MH)⁺

544. Using the compound obtained in Reference Example 28, the followingcompound Examples 20-2 was obtained in the same manner as in Example 19

Example 20-2

545.

546. Yield: 84%

547.¹H-NMR (300 MHz, CDCl₃) δ: 1.25(3H, d), 1.30(9H, s), 2.10(3H, s),2.65-2.67(1H, br m), 3.51(2H, t), 3.60(2H, s), 4.12(2H, s), 5.28(2H, s),7.00(2H, t), 7.16-7.23(5H, m), 7.37-7.42 (3H, m), 7.50-7.53(1H, m),7.61(2H, d), 7.80(2H, d), 7.89(2H, d), 7.94(1H, s), 9.01(1H, s).

548. FAB-Mass m/z 748(MH)⁺

Example 21 Production of3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylaminolphenyl]-4-oxothieno[2,3-b]pyridine

549.

550. A mixture of the compound obtained in Example 19 (0.24 g, 0.33mmol) and 5N sodium hydroxide (0.07 ml) in ethanol (8 ml) was stirred atroom temperature for 1 hour. After the solvent was distilled off underreduced pressure, the residue obtained was dissolved in chloroform (30ml) and the solution was washed with aqueous sodium bicarbonate(saturated, 20 ml) and brine (20 ml). After the organic layer was dried(Na₂SO₄), the solvent was distilled off under reduced pressure and theresidue obtained was recrystallized from chloroform-ether to yield paleyellow powders (0.17 g, 75%).

551. mp 186-188° C.

552.¹H-NMR (300 MHz, CDCl₃) δ: 1.15(2H, dd), 1.44(2H, dd), 2.05(3H, s),3.56(2H, s), 4.01(2H, s), 4.56(1H, br s), 5.24(2H, s), 7.00(2H, t),7.14-7.22(5H, m), 7.41(3H, t), 7.55(2H, d), 7.55(1H, s), 7.70(2H, d),7.88(2H, d), 7.90(1H, s) 8.88(1H, s).

553. Elemental analysis for C₄₀H₃₃N₃O₄SF₂ 1.0H₂O C (%) H (%) N (%)

554. Calculated: 67.88; 4.98; 5.94

555. Found: 67.75; 4.70; 5.90

556. FAB-Mass m/z 690 (MH)⁺

Example 22 Production of3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-(2-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridine

557.

558. Using the compound obtained in Example 20-1, the title compound wasobtained in the same manner as in Example 21.

559. Solvent for recrystallization: chloroform-ether

560. mp 222-223° C.

561.¹H-NMR (300 MHz, CDCl₃) δ: 1.57(6H, s), 2.09(3H, s), 2.50(1H, s),3.59(2H, s), 4.10(2H, s), 5.28(2H, s), 7.01(2H, t), 7.16-7.25(5H, m),7.38-7.44(3H, m), 7.51-7.56(1H, m), 7.67(2H, d), 7.82(2H, d), 7.89(2H,d), 7.93(1H, s), 8.82(1H, s).

562. FAB-Mass m/z 692 (MH)⁺

Example 23 Production of(R)-3-(N-benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(3-hydroxy-2-methylbutyrylamino)phenyl]-4-oxothieno[2,3-b]pyridine

563.

564. Using the compound obtained in Example 20-2, the title compound wasobtained in the same manner as in Example 10.

565. Solvent for recrystallization: chloroform-ether

566. mp 145-146° C.

567.¹H-NMR (300 MHz, CDCl₃) δ: 1.25(3H, d), 2.07(3H, s), 2.63-2.70(1H,m), 2.84(1H, br s), 3.57(2H, s), 3.78-3.81(2H, m), 4.08(2H, s), 5.27(2H,s), 7.00(2H, t), 7.17-7.20(5H, m), 7.37-7.43(3H,m), 7.51-7.56(1H,m),7.61(2H, d), 7.77(2H, d), 7.89(2H, d), 7.94(1H, s), 8.31(1H, br s).

568. FAB-Mass m/z 692 (MH)⁺

Preparation Example 1

569. Using 100 mg of the compound produced in Example 1-2, 165 mg oflactose, 25 mg of corn starch, 4 mg of polyvinyl alcohol and 1 mg ofmagnesium stearate, tablets are produced by a conventional method.

Preparation Example 2

570. The compound produced in Example 1-2 (5 g) is dissolved indistilled water for injection to make a total volume of 100 ml. Thissolution is asepticallyfilteredthrough a 0.22 pm membrane filter(producedby Sumitomo Electric Industries, Ltd. or Sartorius) anddispensed at 2 ml per washed sterile vial, followed by freeze-drying bya conventional method, to yield a 100 mg/vial freeze-dried injectablepreparation.

Preparation Example 3

571. Using 100 mg of the compound produced in Example 3, 165 mg oflactose, 25 mg of corn starch, 4 mg of polyvinyl alcohol and 1 mg ofmagnesium stearate, tablets are produced by a conventional method.

Preparation Example 4

572. The compound produced in Example 3 (5 g) is dissolved in distilledwater for injection to make a total volume of 100 ml. This solution isaseptically filtered through a 0.22 μm membrane filter (produced bySumitomo Electric Industries, Ltd. or Sartorius) and dispensed at 2 mlper washed sterile vial, followed by freeze-drying by a conventionalmethod, to yield a 100 mg/vial freeze-dried injectable preparation.

Preparation Example 5

573.  (1) Compound produced in Example 1-2 5 g  (2) Lactose/crystallinecellulose (particles) 330 g  (3) D-mannitol 29 g  (4) Low-substitutionalhydroxypropyl cellulose 20 g  (5) Talc 25 g  (6) Hydroxypropyl cellulose50 g  (7) Aspartame 3 g  (8) Dipotassium glycyrrhizinate 3 g  (9)Hydroxypropylmethyl cellulose 2910 30 g (10) Titanium oxide 3.5 g (11)Yellow iron sesquioxide 0.5 g (12) Light silicic anhydride 1 g

574. Components (1), (3), (4), (5), (6), (7) and (8) are suspended ordissolved in purified water and coated on the core particles (2) toyield base fine subtilae, which are then further coated with components(9) through (11) to yield coated fine subtilae, which are then mixedwith component (12) to yield 500 g of 1% fine subtilae of the compound.These subtilae are divided to 500 mg folded subtilae.

Experimental Example 1

575. Preparation of ¹²⁵I-leuprorelin

576. To a tube containing 10 μl of a 3×10⁻⁴M aqueous solution ofleuprorelin and 10 μl of 0.01 mg/ml lactoperoxidase, 10 μl (37 MBq)ofasolution of Na¹²⁵I was added. After stirring, 10 μl of 0.001% H₂O₂was added, and a reaction was carried out at room temperature for 20minutes. By adding 700 μl of a 0.05% TFA (trifluoroacetic acid)solution, the reaction was stopped, followed by purification byreversed-phase HPLC. The HPLC conditions used are shown below.¹²⁵I-leuprorelin was eluted at a retention time of 26 to 27 minutes.

577. Column: TSKgel ODS-_(80™) (TM indicates a registered trademark; thesame applies below) CTR (4.6 mm×10 cm)

578. Eluents: Solvent A (0.05% TFA)

579. Solvent B (40% CH₃CN-0.05% TFA)

580. 0 minute (100% Solvent A)−3 minutes (100% Solvent A)−7 minutes (50%Solvent A+50% Solvent B)−40 minutes (100% Solvent B)

581. Eluting temperature: Room temperature

582. Elution rate: 1 ml/min

Experimental Example 2

583. Preparation of a rat pituitary anterior lobe membrane fractioncontaining GnRH receptors

584. Anterior lobes of the pituitary glands were isolated from fortyWistar rats (8 weeks old, male), and washed with ice-cooled homogenatebuffer [25 mM Tris (tris(hydroxymethyl)aminomethane)-HCl, 0.3M sucrose,1 mM EGTA (glycol-etherdiamine-N,N,N′,N′-tetraacetic acid), 0.25 mM PMSF(phenylmethylsulfonyl fluoride), 10 U/ml aprotinin, 1 mg/ml pepstatin,20 mg/ml leupeptin, 100 mg/ml phosphoramidon, 0.03% sodium azide, pH7.5]. The pituitary tissue was floated in 2 ml of the homogenate bufferand homogenized using a Polytron homogenizer. The homogenate wascentrifuged at 700×g for 15 minutes. The supernatant was taken in anultracentrifuge tube and centrifuged at 100, 000×g for 1 hour to providea membrane fraction pellet. This pellet was suspended in 2 ml of assaybuffer [25 mM Tris-HCl, 1 mM EDTA (ethylenediaminetetraacetic acid),0.1% BSA (bovine serum albumin), 0.25 mM PMSF, 1 mg/ml pepstatin, 20mg/ml leupeptin, 100 mg/ml phosphoramidon, 0.03% sodium azide, pH 7.5)and the suspension was centrifuged at 100,000×g for 1 hour. The membranefraction recovered as a pellet was resuspended in 10 ml of assay buffer,divided into portions, preserved at −80° C. and thawed when needed.

Experimental Example 3

585. Preparation of CHO (Chinese hamster ovarian) cell membrane fractioncontaining human GnRH receptor

586. Human GnRH receptor-expressing CHO cells (EP-A-678577) (10⁹ cells)were suspended in phosphate-buffered saline supplemented with 5 mM EDTA(ethylenediaminetetraacetic acid) (PBS-EDTA) and centrifuged at 100×gfor 5 minutes. To the cell pellet, 10 ml of a cell homogenate buffer (10mM NaHCO₃, 5 mM EDTA, pH 7.5) was added, followed by homogenizationusing the Polytron homogenizer. After centrifugation at 400×g for 15minutes, the supernatant was transferred to an ultracentrifugation tubeand centrifuged at 100,000×g for 1 hour to yield a membrane fractionprecipitate. This precipitate was suspended in 2 ml of an assay bufferand centrifuged at 100,000×g for 1 hour. The membrane fraction recoveredas a precipitate was again suspended in 20 ml of the assay buffer,dispensed, and stored at −80° C. before use upon thawing.

Experimental Example 4

587. Determination of ¹²⁵I-leuprorelin binding inhibition rate

588. The rat and human membrane fractions prepared in ExperimentalExamples 2 and 3 were diluted with the assay buffer to yield a 200 mg/mldilution, which was then dispensed at 188 ml per tube. Where the ratpituitary anterior lobe membrane fraction was used, to each tube, 2 mlof a solution of 0.1 mM compound in 60% DMSO (dimethyl sulfoxide) and 10μl of 38 nM ¹²⁵I-leuprorelin were added simultaneously. Where the cellmembrane fraction of the CHO with human GnRH receptors expressed, toeach tube, 2 ml of a solution of 2 mM compound in 60% DMSO and 10 μl of38 nM ¹²⁵I-leuprorelin were added simultaneously. To determine maximumbinding quantity, a reaction mixture of 2 μl of 60% DMSO and 10 μl of 38nM ¹²⁵I-leuprorelin was prepared. To determine non-specific bindingamount, a reaction mixture of 2 μl of 100 μM leuprorelin in solution in60% DMSO and 10 μl of 38 nM ¹²⁵I-leuprorelin was prepared.

589. Where the rat or bovine pituitary anterior lobe membrane fractionwas used, the reaction was conducted at 4° C. for 90 minutes. Where themembrane fraction of the CHO with human GnRH receptors expressed wasused, the reaction was carried out at 25° C. for 60 minutes. After eachreaction, the reaction mixture was aspirated and filtered through apolyethyleneimine-treated Whatman glass filter (GF-F). After thisfiltration, the radioactivity of ¹²⁵-leuprorelin remaining on the filterpaper was measured with a γ-counter. The expression (TB-SB)/(TB-NSB)×100(where SB= radioactivity with the compound added, TB=maximum boundradioactivity, NSB=nonspecifically bound radioactivity) was calculatedto find the binding inhibition rate (%) of each test compound.Furthermore, the inhibition rate was determined by varying theconcentration of the test substance and the 50% inhibitory concentration(IC₅₀ value) of the compound was calculated from Hill plot. The resultsare shown in below. binding inhibition IC₅₀ value rate (%) (μM) TestCompound Rat (1 μM) Human (20 μM) Rat Human Compd. of Ex. 1-2 96 1020.06 0.0001 Compd. of Ex. 3 62 NT 0.6  0.0002

Experimental Example 5

590. Suppression of blood LH in castrated monkeys

591. The compound produced in Example 1-2 was orally administered tocastrated male cynomolgus monkeys (Macaca fascicularis), and blood LHwas quantified. The male cynomolgus monkeys, used at 3 years 8 months to7 years 7 months of age at time of experimentation, had been castratedmore than half a year prior to the examination. Test animals [n =2;compound (1) and compound (2)] were given 30 mg/kg (3 ml/kg) of thecompound suspended in 0.5% methyl cellulose at a final concentration of1% by oral administration, and control animals [n=3; control (1),control (2) and control (3)] were given 3 ml/kg of the 0.5% methylcellulose dispersant alone by oral administration. At 24 hours andimmediately before administration and at 2, 4, 6, 8, 24, and 48 hoursafter administration, blood was collected for heparinized plasma samplesvia the femoral vein and immediately stored under freezing conditions.

592. Plasma LH concentrations were determined by a bioassay using mousetesticular cells. The testicular cells were collected frommale BALB/cmice (8 to 9 weeks of age) and washed three times with 1 ml ofDulbecco's modified Eagle medium (DMEM-H) containing 20 mM HEPES and0.2% BSA per testis. After incubation at 37° C. for 1 hour, the cellswere passed through a nylon mesh filter (70 pm) and dispensed to testtubes at 8×10⁵ cells/tube. After the cells were washed twice with 0.4 mlof DMEM-H, 0.4 ml of a DMEM-H solution containing either equine LH(Sigma Company), as the standard LH, or monkey plasma, previouslydiluted up to 1,000 fold, as the test sample, was added, followed by areaction at 37° C. for 2 hours. The testosterone concentration in theculture supernatant was determined by a radioimmunoassay (CISDiagnostics Company), and the LH concentration in the test monkey plasmawas calculated from the standard curve for the standard equine LH.

593. The results are given together in FIG. 1.

594. The compound indicated is the compound obtained in Example 1-2.

595. For the control (1), the control (2), and the control (3), changesover time in the LH concentrations in each animal are expressed aspercent ratios to the respective reference values, which comprise the LHconcentrations immediately before administration in each control animal(cynomolgus monkey). Similarly, for the compound (1) and the compound(2), changes over time in the LH concentrations in each animal(cynomolgus monkey) receiving the compound of Example 1-2 are expressedas percent ratios to the respective reference values, which comprise theLH concentrations immediately before administration of the compound. Thetime of administration was defined as zero (0), and hours before andafter administration shown by minus and plus, respectively.

INDUSTRIAL APPLICABILITY

596. The compound of the present invention possesses excellentgonadotropin-releasing hormone antagonizing activity. It is also good inoral absorbabilityandexcellent in stability and pharmacokinetics. Withlow toxicity, it is also excellent in safety.

597. The compound of the present invention can therefore be used as aprophylactic or therapeutic agent for hormone-dependent diseases etc.Specifically, it is effective as a prophylactic or therapeutic agent forsex hormone-dependent cancers (e.g., prostatic cancer, uterine cancer,breast cancer, pituitary tumor), prostatic hypertrophy, hysteromyoma,endometriosis, precocious puberty, amenorrhea syndrome, multilocularovary syndrome, pimples etc, or as a pregnancy regulator (e.g.,contraceptive), infertility remedy or menstruation regulator. It is alsoeffective as an animal estrous regulator, food meat quality improvingagent or animal growth regulator in the field of animal husbandry, andas a fish spawning promoter in the field of fishery.

1. A compound of the formula:

wherein R¹ represents a C₁₋₇ alkyl group which may be substituted, aC₃₋₇ cycloalkyl group which may be substituted, a C₁₋₆ alkoxyamino groupwhich may be substituted or a hydroxyamino group which may besubstituted; and R² represents a C₁₋₇ alkyl group which may besubstituted or a phenyl group which may be substituted; when R¹ is anunsubstituted C₁₋₇ alkyl group, then R² is a substituted C₁₋₇ alkylgroup or a substituted phenyl group, or a salt thereof.
 2. A compound ofclaim 1 or a salt thereof, wherein R¹ is (1) a C₁₋₇ alkyl group whichmay be substituted by 1 to 5 substituents selected from the groupconsisting of (i) hydroxy, (ii) C₁₋₇ acyloxy, (iii) amino which may besubstituted by 1 or 2 substituents selected from the group consisting ofC₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyland C₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy which may be substituted by 1 to 3substituents selected from the group consisting of C₃₋₇cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆ alkoxy-carbonyl, (2)a C₃₋₇ cycloalkyl group which may be substituted by 1 to 3 substituentsselected from the group consisting of (i) hydroxy, (ii) C₁₋₇ acyloxy,(iii) amino which may be substituted by 1 or 2 substituents selectedfrom the group consisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl,C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy whichmay be substituted by 1 to 3 substituents selected from the groupconsisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆alkoxy-carbonyl, (3) a C₁₋₆ alkoxyamino group which may be substitutedby 1 to 5 substituents selected from the group consisting of (i)hydroxy, (ii) C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or2 substituents selected from the group consisting of C₁₋₆alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl andC₁₋₃ alkyl, (iv) C₁₋₁₀ alkoxy which may be substituted by 1 to 3substituents selected from the group consisting of C₃₋₇cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (v) C₁₋₆ alkoxy-carbonyl, or(4) a hydroxyamino group which may be substituted by 1 or 2 substituentsselected from the group consisting of (i) C₁₋₇ acyloxy, (ii) amino whichmay be substituted by 1 or 2 substituents selected from the groupconsisting of C₁₋₆ alkoxy-carbonyl, benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃alkylsulfonyl and C₁₋₃ alkyl, (iii) C₁₋₁₀ alkoxy which may besubstituted by 1 to 3 substituents selected from the group consisting ofC₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxy and (iv) C₁₋₆ alkyl; and R²is (1) a C₁₋₇ alkyl group which may be substituted by 1 to 5substituents selected from the group consisting of (i) hydroxy, (ii)C₁₋₇ acyloxy, (iii) amino which may be substituted by 1 or 2substituents selected from the group consisting of C₁₋₆ alkoxy-carbonyl,benzyloxycarbonyl, C₁₋₃ acyl, C₁₋₃ alkylsulfonyl and C₁₋₃ alkyl, (iv)C₁₋₁₀ alkoxy which may be substituted by 1 to 3 substituents selectedfrom the group consisting of C₃₋₇ cycloalkyloxy-carbonyl and C₁₋₃ alkoxyand (v) C₁₋₆ alkoxy-carbonyl, or (2) a phenyl group which may besubstituted by 1 to 5 substituents selected from the group consisting ofhalogen, C₁₋₃ alkyl and C₁₋₃ alkoxy.
 3. A compound of claim 1 or a saltthereof, wherein R¹ is a C₁₋₇ alkyl group which may be substituted or aC₃₋₇ cycloalkyl group which may be substituted.
 4. A compound of claim 1or a salt thereof, wherein R¹ is a substituted branched C₃₋₇ alkyl groupor a substituted C₃₋₇ cycloalkyl group.
 5. A compound of claim 1 or asalt thereof, wherein R¹ is a C₁₋₇ alkyl group substituted by hydroxy ora C₃₋₇ cycloalkyl group substituted by hydroxy.
 6. A compound of claim 1or a salt thereof, wherein R¹ is a substituted C₃₋₇ cycloalkyl group. 7.A compound of claim 1 or a salt thereof, wherein R¹ is a cyclopropylgroup which may be substituted by hydroxy.
 8. A compound of claim 1 or asalt thereof, wherein R² is a branched C₃₋₇ alkyl group which may besubstituted.
 9. A compound of claim 1 or a salt thereof, wherein R² is aphenyl group which may be substituted.
 10. A compound of claim 1 or asalt thereof, wherein R² is a phenyl group.
 11. A compound of claim 1 ora salt thereof, wherein R¹ is a C₃₋₇ cycloalkyl group and R² is a C₁₋₆alkyl group.
 12. A compound of claim 1 or a salt thereof, wherein R¹ is(1) a C₁₋₄ alkyl group substituted by 1 or 2 hydroxy, (2) a C₃₋₇cycloalkyl group substituted by hydroxy, or (3) a C₁₋₃ alkoxyaminogroup; and R² is an isopropyl group or a phenyl group.
 13. A compound ofclaim 1 or a salt thereof, wherein R¹ is (1) a C₁₋₇ alkyl group whichmay be substituted by 1 or 2 substituents selected from the groupconsisting of hydroxy, C₁₋₃ alkyl-carbonyloxy, amino,benzyloxycarbonylamino, C₁₋₃ alkoxy, C₁₋₃ alkoxy-C₁₋₃ alkoxy and C₁₋₃alkoxy-carbonyl, (2) a C₃₋₇ cycloalkyl group which may be substituted bya hydroxy or a C₁₋₃ alkyl-carbonyloxy, or (3) a C₁₋₃ alkoxyamino group;and R² is (1) an isopropyl group which may be substituted by a hydroxyor (2) a phenyl group. 14.3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-(4-cyclopropanecarbonylaminophenyl)-4-oxothieno[2,3-b]pyridineor a salt thereof. 15.3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-(3-hydroxy-2-methylpropionylamino)phenyl]-4-oxothieno[2,3-b]pyridineor a salt thereof. 16.3-(N-Benzyl-N-methylaminomethyl)-4,7-dihydro-5-isobutyryl-7-(2,6-difluorobenzyl)-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine or a salt thereof. 17.3-(N-Benzyl-N-methylaminomethyl)-5-benzoyl-7-(2,6-difluorobenzyl)-4,7-dihydro-2-[4-[(1-hydroxycyclopropyl)carbonylamino]phenyl]-4-oxothieno[2,3-b]pyridine or a salt thereof.
 18. A process forproducing a compound of claim 1 or a salt thereof, which comprisesreacting a compound of the formula:

wherein R² represents a C₁₋₇ alkyl group which may be substituted or aphenyl group which may be substituted, or a salt thereof, with i) acompound of the formula: R^(1a)COOHwherein R^(1a) represents a C₁₋₇alkyl group which may be substituted or a C₃₋₇ cycloalkyl group whichmay be substituted, or a salt thereof or a reactive derivative thereof;or ii) carbonyldiimidazole, phosgene or a chloroformate, followed byreacting with a compound of the formula: R^(1b)Hwherein R^(1b)represents a C₁₋₆ alkoxyamino group which may be substituted or ahydroxyamino group which may be substituted, or a salt thereof.
 19. Aprocess for producing a compound of claim 3 or a salt thereof, whichcomprises reacting a compound of the formula:

wherein R² represents a C₁₋₇ alkyl group which may be substituted or aphenyl group which may be substituted, or a salt thereof, with acompound of the formula: R^(1a)COOH wherein R^(1a) represents a C₁₋₇alkyl group which may be substituted or a C₃₋₇ cycloalkyl group whichmay be substituted, or a salt thereof or a reactive derivative thereof.20. A pharmaceutical composition which comprises a compound of claim 1or a salt thereof.
 21. A pharmaceutical composition of claim 20 which isfor antagonizing gonadotropin-releasing hormone.
 22. A pharmaceuticalcomposition of claim 21 which is for preventing or treating a sexhormone dependent disease.
 23. A method for antagonizinggonadotropin-releasing hormone in a mammal in need thereof whichcomprises administering to said mammal an effective amount of a compoundof claim 1 or a salt thereof with a pharmaceutically acceptableexcipient, carrier or diluent.
 24. Use of a compound of claim 1 or asalt thereof for manufacturing a pharmaceutical composition forantagonizing gonadotropin-releasing hormone.